Biotech Crunch

DNA Sequencing using the Maxam-Gilbert Method

2012-01-21T10:53:00.001+05:00

Maxam-Gilbert Sequencing With this method of DNA sequencing instead of synthesizing DNA in vitro and stopping the synthesis reactions with chain terminators, this method starts with full-length, end labeled DNA and cleaves it with base specific reagents. So how this method works with guanine bases, but the same principle applies to all four bases; First we end-label a DNA fragment we want to sequence.

This can be 5’- or 3’-end labeling. Next, we modify one kind of base. Here we use dimethyl sulfate (DMS) to methylate guanines. (Actually, this reagent also methylates adenines, but not in a way that leads to DNA strand cleavage.) As in the chain termination method, we do not want to affect every guanine, or we will produce only tiny fragments that will not allow us to determine the DNA’s sequence. Therefore, we do the methylation under mild conditions that lead to an average of only one methylated guanine per DNA strand. Next, we use a reagent (piperidine) that does two things: it causes loss of the methylated base, then it breaks the DNA backbone at the site of the lost base (the apurinic site). In this case, the G in the middle of the sequence was methylated, so strand breakage occurred there, producing a labeled trimer.

In another DNA molecule, the first G could not be methylated, giving rise to a labeled phosphate (the base and sugar would be lost in the chemical cleavages). Finally, we electrophorese the products and detect them by autoradiography, just as in the chain-termination method. Of course, we need to run three other reactions that cleave at the other three bases. There are several ways of doing this. For example, we can weaken the glycoside bonds to both adenine and guanine with acid; then piperidine will cause depurination and strand breakage after both As and Gs. If we electrophorese this A + G reaction beside the G only reaction, we can obtain the As by comparison. Similarly, hydrazine opens both thymine and cytosine rings, and piperidine can then remove these bases and break the DNA strand at the resulting apyrumidine sites. In the presence of 1M NaCl, hydrazine is specific for cytosine only, so we can run this reaction next to the C+T reaction and obtain the Ts by comparison.

Choosing the right DNA polymerase for your PCR

2012-01-10T12:43:00.002+05:00

Success of the polymerase chain reaction (PCR) largely depends on the choice of the appropriate DNA polymerase. DNA polymerase is one of the major components needed for setting up a PCR. For PCR, a thermo-stable DNA polymerase is essential, so that it can endure higher temperatures during the cycling conditions. Therefore, thermo-stable DNA polymerases serve as a key player in the current methods of DNA amplification and sequencing.

Based on their amino acid sequences, DNA polymerases are categorized into six families: A, B, C, D, X and Y. Thermophilic enzymes are found in all the six families. Same reaction is catalyzed by all DNA polymerases, that is, adding nucleotides to the 3′-end of the DNA primer to synthesize the new DNA strand complementary to the template DNA. The thermo-stable DNA polymerases synthesize DNA in a template-directed manner, and require primer-template hybrid to begin the synthesis.

Development of the PCR resulted in advancement of countless molecular biology techniques. Following PCR, a large number of various subsequent experimentation can be done. PCR for different purposes require different types of DNA polymerases. PCR is generally done for screening of the recombinant clone or for just simply determining the size of a particular DNA fragment. This can be carried out with simple DNA polymerases. But for cloning the DNA fragment for its expression, library construction, genome walking, RACE (Rapid Amplification of cDNA Ends) etc., different kinds of DNA polymerases are required.

Therefore, selecting an appropriate DNA polymerase, in accordance to the application, is extremely significant for the success of the experiment. DNA polymerases possess following four basic properties on the basis of which suitable DNA polymerase can be selected:

· Thermosatability

For the amplification procedures, DNA polymerases should be thermo-stable essentially. During each cycle of the PCR reaction, a denaturation step (at approximately 95°C) is there to separate two strands of the DNA molecule for next round of synthesis. Therefore, DNA polymerase should be durable enough to tolerate such a high temperature without losing its activity.

Half-life of a DNA polymerase at a specific temperature determines its thermostability. Its survival in a particular procedure largely depends on the factors like reaction mixture, protein concentrations and other reaction conditions.

· Extension rate

The number of dNTPs added per second per molecule of DNA polymerase is known as the extension rate. It highly depends on the reaction mixture and DNA templates. Sometimes there is formation of structures in the DNA molecules, as a result of which primer elongation ceases.

Generally, higher extension rates of DNA polymerases are desired. This facilitates amplification of longer DNA fragments.

· Processivity

Processivity is the probability that a DNA polymerase will not detach from the DNA after the attachment of a nucleotide, while translocating to the next position. It indicates the average number of the nucleotides that a DNA polymerase adds in a single binding event.

Similar to extension rate, processivity depends on the components of the reaction mixture and the sequence of the DNA template. On heterogeneous templates, processivity of each template position depends on the salt concentration.

· Fidelity

Fidelity denotes the frequency of insertion of the correct nucleotide per incorrect insertion. It actually refers to the ability of the DNA polymerases to insert correct nucleotides. It is an intrinsic property of the DNA polymerases. Therefore, DNA polymerases having low efficiency of correct nucleotide insertion, i.e. inefficient DNA polymerases, exhibit low fidelity, whereas, efficient polymerases exhibit high fidelity.

On the basis of the above mentioned properties, different types of DNA polymerases are available commercially. In accordance to the requirements and experiments, appropriate DNA polymerase can be selected from the following classes:

1) Taq DNA polymerase

Taq DNA polymerase was purified from bacterium Thermus aquaticus, found in hot springs. It is the most commonly used thermophilic DNA polymerase that catalyzes template-directed synthesis of DNA using nucleotide triphosphates.

It is used for general purpose PCRs, such as colony PCR (for screening of the recombinant clones), amplification of the DNA fragment for estimating its size, simple detection of the amplified product, PCR based molecular marker studies, etc.

They generally produce DNA fragments with ‘A’ overhang at 3’-end. This means that whenever a DNA fragment will be amplified with Taq DNA polymerase, the amplified products will have a single adenine base at 3’-terminal. Thus, the amplified DNA fragment can be directly cloned into T/A cloning vector.

Properties

· Taq polymerase possesses maximum catalytic activity at 75-80°C. It has half-life of 1.6 hours at 95°C.

· It has a extension rate of 1kb/minute.

· It has been observed that Taq polymerase dissociates from the DNA after attaching approximately 40 nucleotides,. Hence, exhibiting a good processivity.

· It lacks 3’ to 5’ exonuclease activity. As a result of this, it generally has higher error rate and less fidelity. It has error rate between 1X10^-4 to 2X10^-5 errors per base pair.

Limitations

Since Taq polymerases have higher error rate, it cannot be used for amplification of DNA fragments where high accuracy is desired. For example, it cannot be used for amplifying the DNA fragments to be cloned and expressed, and for mutagenesis studies.

2) Proofreading DNA polymerases

DNA polymerases are said to be proofreading when they possess 3’ to 5’ exonuclease activity. Whenever there in incorporation of non-complementary nucleotide in the growing DNA strand, the proofreading DNA polymerase by the virtue of its 3’ to 5’ exonuclease activity, removes the erroneously attached bases by hydrolysis. This is an irreversible reaction. It significantly, increases the accuracy of the DNA synthesis from the template DNA, thereby exhibiting high fidelity.

Because of high fidelity, they are extremely useful for techniques that demand high accuracy in the DNA synthesis. For instance, for cloning and expression of amplified product, mutagenesis studies, etc. proofreading enzymes are obvious enzymes to choose.

They usually generate blunt-ended PCR products. Therefore, PCR fragments amplified by proofreading polymerases can be directly used for blunt-end cloning.

Properties

· Exhibits maximum catalytic activity at 68-75°C and have superior thermostability. Their half-life is 6.7 hours at 95°C.

· Relatively slower than Taq polymerase. Extension rate is approximately 0.5kb/minute.

· Their processivity is quite low as compared to Taq polymerase, i.e. approximately 4-30 nucleotides.

· Possess 3’ to 5’ exonuclease proofreading activity. They have far less errors as compared to Taqpolymerase. Their error rate is approximately 1.5 X 10^-6error per base pair. This provides 5-15 fold higher fidelity than Taq polymerase.

Limitations

They are quite slower and therefore require more time to amplify the DNA fragments. Since they have low processivity, very high optimization is required. For this, generally gradient PCR is done.

Example

· Pfu DNA polymerase (Stratagene).

· Vent_R® DNA polymerase (New England Biolabs).

3) Polymerases for the amplification of long templates

Generally, Taq polymerase is able to amplify DNA fragment of ~3kb only. Whereas, the proofreading DNA polymerases can amplify PCR products of size up to ~6kb. Therefore, in order to amplify DNA fragments of much larger size, several DNA polymerases have been developed. Generally, they are mixture of a Taqpolymerase and a proofreading polymerase.

They are extremely useful for producing high yield of PCR product from genomic DNA with accuracy. They can amplify fragments as large as 30kb in size.

Properties

· Highly thermostable similar to Taq polymerase. Optimum temperature is usually 68°C.

· Extension rate is little bit higher than Taq polymerase, i.e. ~1.5kb/minute.

· Processivity similar to Taq polymerase.

· Due to inherent 3’ to 5’ exonuclease proofreading activity, they are 3-fold more accurate than Taqpolymerase.

Limitations

Since it is a mixture of Taq polymerase and proofreading DNA polymerase, its fidelity is not very high.

Examples

· Expand long template PCR system (Roche)

· LongAmp® Taq DNA polymerase (New England Biolabs).

4) Hot start polymerases

Hot start PCR is a modification of conventional PCR. It is used mainly to suppress non-specific product amplification and to increase the yield of the desired product.

In conventional PCR, the Taq polymerase remains active at room temperature and even on ice, to some extent. At this point, when all reaction components are mixed, primers can anneal non-specifically to the template DNA. The non-specific annealed primers can be extended by Taq polymerase, resulting in accumulation of non-specific products, thereby decreasing the yield of desired product.

Therefore, in hot start PCR, the DNA polymerase is kept inactive with the help of neutralizing monoclonal antibody until higher temperatures are reached. When the temperature raises, the antibody dissociates from the enzyme and gets inactivated making the DNA polymerase active, it considerably reduces non-specific priming, primer-dimer formation and, thus, increases the product yield.

They are very useful when the amount of DNA template is very low (i.e. less than 10⁴ copies of template DNA), when the DNA template exhibits high complexity (e.g. mammalian genomic DNA), and when several pairs of primers are there in the PCR (e.g. multiplex PCR). In all such cases, hot start PCR works best in combination with ‘touchdown PCR’ protocol.

Properties

· Remains inactive until higher temperatures are attained.

· Their extension rate and processivity is similar to that of Taq polymerase, since they are basically Taqpolymerase bound with an antibody.

· Lacks 3’ to 5’ exonuclease proofreading activity. Some suppliers provide blend of proofreading polymerase along with it to provide 3’ to 5’ exonuclease activity.

Limitations

Have lower fidelity and thus higher error rate.

Examples

· Advantage® 2 PCR enzyme system (Clontech).

· AccuStart^TM Taq DNA polymerase (Quanta Biosciences).

2) Next Generation Polymerases

Next generation of polymerases have been developed to overcome the shortcomings of the existing proofreading polymerases. They are engineered and generally are Pfu-based DNA polymerases. They are incorporated with several characteristics that provide them increased processivity, high fidelity and extreme speed.

By the use of high affinity DNA binding domain, their processivity has been increased dramatically. With the help of this domain, DNA polymerase can anchor much better. This prevents its early dissociation from the template DNA. Therefore, there is incorporation of more nucleotides per binding event due to improved processivity. This enhances PCR yield and shortens the extension time.

They are very suitable for high performance cloning in very short time. Their improved speed makes them desirable for higher throughput. They also provide very high yield of the PCR product. They are ideal for difficult targets and provide accurate results even with complex DNA templates having very high GC content (i.e. >80%). They can amplify DNA fragments up to ~10kb. They are highly sensitive and can use very low amounts of DNA for amplification.

Properties

· Highly thermostable, as developed from Pfu polymerases.

· Modified to generate PCR fragments in much shorter extension times. With some next generation DNA polymerases, extension rate is as high as 1Kb/15-30second.

· Their processivity is 12-fold higher than the Pfu DNA polymerases.

· Highly accurate. Exhibits robust performance and reliability.

Limitations

They are relatively expensive.

Examples

· Herculase® II Fusion DNA polymerase (Stratagene).

· Phusion® DNA polymerase (Finnzymes).

Hence, from above discussion it is clear that for routine and general PCR you should use Taq DNA polymerase, whereas, if you have to express a gene or carry out mutagenesis experiment, you should go for the proofreading or new generation DNA polymerases. Similarly, if you need to carry out RACE or Genome Walking experiments, a Hotstart polymerase should be your pick. In addition, if you want to amplify large template then you should choose Expand Long DNA polymerase.

PCR Master Mix

2012-01-10T12:36:00.000+05:00

The polymerase chain reaction (PCR) is used to amplify a specific fragment of DNA strand from a complex mixture of initial starting material. For setting up a PCR, you need to prepare a master mix. Generally, the PCR master mix consists of a target double-stranded DNA template, two oligonucleotide primers which hybridize to bordering sequences on either strands of the template, all four deoxyribonucleoside triphosphates (dNTPs) and a DNA polymerase.

Generally, you need to prepare 10-200µl reaction volume in small reaction tubes (200-500µl volume tubes) for setting up a PCR. The PCR is carried out in a thermal-cycler. Thermal-cycler is a machine that provides varying temperatures by heating or cooling the reaction tubes, as per the requirement of each reaction step. It is preferable if you use thin-walled reaction tubes, since it facilitates rapid thermal equilibration by maintaining proper thermal conductivity.

Now-a-days, in most of the thermal-cyclers, heated lids are there. As a result of this, the evaporating reaction mixture does not condense at the top of the reaction tube and remain within the reaction volume, maintaining the original composition. In thermal-cyclers without heated lids, a layer of mineral oil is added on the top of the reaction mixture to prevent evaporation. Alternatively, a wax ball can be inserted inside the reaction tube.

Once you have selected the appropriate target substrate, you need the following basic components for setting up the reaction

1) Target DNA

You can obtain DNA for initial amplification from different types of sources. The template DNA source can be plasmid DNA, genomic DNA, cDNA, prokaryotic cells or even eukaryotic cells. You just need to simply boil prokaryotic or eukaryotic cell samples for extracting the DNA for PCR.

The minimal amount of template DNA required for PCR depends on the source. For example, 1µg of DNA is required if isolated from mammalian cells, whereas 1ng of DNA is sufficient enough if the source is bacteria. However, in case of plasmid DNA, you just need as little as 1pg of DNA for PCR amplification.

Double-stranded DNA (e.g. plasmid, genomic DNA etc.) as well as single-stranded DNA (e.g. cDNA) can be used as template DNA. Amplifications can be obtained both from circular-coiled DNA and linear DNA. However, it has been found that amplification from circular-coiled DNA is less efficient as compared to linear DNA. The main reason behind this is the huge and complex structure of circular-coiled DNA, which hinders in the proper binding of the primers to the template DNA, resulting in poor amplification. With most of the common PCR methods, DNA fragments of up to ~10kb can be easily amplified. However, using specialized techniques even the fragments up to 40kb can be amplified.

2) Primers

The most crucial factor that decides the efficiency and specificity of the PCR amplification is the primers. Primers are the short stretches of oligonucleotides that anneals to the template DNA to amplify the fragment. They are essential for PCR amplifications because DNA polymerases can only bind the new nucleotides to the existing oligonucleotide/strand in 5’ to 3’ direction. Thus, primer hybridized to the template DNA serves as a short DNA strand to which the DNA polymerases can easily keep on adding new nucleotides to copy the existing DNA strand.

The primers are designed complementary to the DNA sequences bordering the target sequence to be amplified. But at the same time you must check that the forward and reverse primers are not complementary to each other, otherwise they will bind to each other to form primer dimers. This will obstruct proper DNA amplification, since no primer population will be left to anneal to the template DNA.

The other parameters that you need to keep in your mind while designing your primers are primer length, GC content and the melting temperatures. The ideal primer length is 20-28mer. The appropriate GC content is 40-60% and you should try to keep melting temperature in the range of 55-65ºC. There are many bioinformatics tools available with the help of which you can design your primers. Some examples are Oligo calculator, Generunner, Primer3, GeneFisher etc.

While designing primers the three objectives that you need to keep in your mind are:

i) The primers must be designed in such a way that there is high yield of the desired product.

ii) Amplification of unwanted non-specific sequences should be avoided.

iii) Subsequent manipulation of the amplified product shall be achieved easily.

3) dNTPs

Equimolar concentrations of dATP, dTTP, dCTP and dGTP must be present in the standard PCR. If you are using 1.5mM MgCl₂along with the Taq DNA polymerase in 50µl reaction, then you should add 200-250µM of each dNTP. Higher concentrations of dNTPs should be avoided, since it reduces the yield by quenching the Mg²⁺ions which are essential for proper activity of polymerases. Also, there will be more probability of incorporation of mismatched nucleotides by the polymerase.

Now-a-days, stocks of varying concentrations (10, 25 or 100mM) of dNTPs are available commercially. You should store stocks of these dNTPs at -20ºC. These stocks are provided with pH 8.1, which minimizes its damage from freeze and thaw. However, it would be preferable if you store these stocks in small aliquots, as this will prevent dNTPs from degradation by repeated freezing and thawing.

4) DNA polymerase

DNA polymerase is an enzyme needed for the synthesis of the DNA fragment. For PCR, a thermo-stable DNA polymerase is required, so that it can withstand higher temperatures. DNA polymerase needs a short stretch of oligonucleotides for the synthesis of new DNA fragment.

The polymerase gets attached to the primer-template hybrid and synthesis of new DNA strand begins using primers as a starting point. It keeps on adding single free nucleotides one by one to the exposed 3′-hydroxyl group provided by the primer. The DNA synthesis occurs in 5′ to 3′ direction only and not in 3′ to 5′ direction. This is because the nucleotides can be added only to the 3′ end and not to the 5′ end of the nucleic acid by DNA polymerase.

Now-a-days, a wide range of enzymes are available commercially. You can choose among them on the basis of their reliability, efficiency and ability to synthesize large fragments in accordance to your needs. IfTaq polymerase is used, then generally its 2-5 units are added in 50µl reaction volume. If higher amount of enzyme than this level is taken, then it may lead to accumulation of non-specifically amplified PCR product and there will be low yield of desired fragment. If accurate and flawless amplification of the DNA fragment is desired, then proofreading DNA polymerases are also available. For Example, Pfu DNA polymerase, Vent DNA polymerase, Phusion DNA Polymerase, etc. They remove mismatched nucleotides, if any, during amplification by their 3′ to 5′ exonuclease activity.

5) Standard Reaction Buffer

Buffer is the most basic component of the reaction master mix. It provides the platform for the reaction to take place. The main function of the buffer is to maintain the pH so as to make the reaction feasible. Any change in the PCR buffer will affect the consequence of the amplification. Basically, standard buffer consists of Tris-Cl at a concentration of 10mM.

During PCR cycling, when the temperature reaches 72ºC, there is a dip of more than a full unit in the pH of the reaction mixture. The optimal pH for the reaction mixture is ~7.2. Therefore, the pH of the standard reaction buffer is kept approximately 8.3-8.8, so that during the cycling process the pH of the reaction mixture may become desirable after getting reduced.

Buffer also contains KCl and MgCl₂ as the source of monovalent and divalent cations.

i) Mono-valent Cations

You need appropriate amount of mono-valent cations in the standard reaction buffer for primer annealing and for proper amplification of the DNA fragments. Generally, KCl is added as the source of mono-valent cations. Generally, 50mM KCl is sufficient enough to amplify larger DNA fragments. However, if the target DNA fragment is shorter, then higher concentration of KCl (70-100mM) would be suitable for the reaction.

ii) Di-valent Cations

Presence of free divalent cations plays a critical role in exhibiting proper activity by all thermo-stable DNA polymerases. You will find MgCl₂ as the most common source of divalent cations in most of the commercially available standard reaction buffers. Sometimes MnCl₂ is used alternatively but polymerases work less efficiently in this case. Generally, 1.5mM MgCl₂ is used in standard reaction buffers. Its higher concentration results in the production of non-specific amplified products, whereas inadequate amount reduces the yield.

Now-a-days, stock solutions of buffers are supplied along with the DNA polymerase. These buffers are optimized for particular type of DNA polymerase. For example, Taq polymerase is supplied with 10X standard Taq buffer. Similarly, different DNA polymerases are supplied with their respective buffers.

PCR : Polymerase Chain Reaction

2012-01-06T22:09:00.000+05:00

Polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

PCR principles and procedure

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

DNA template that contains the DNA region (target) to be amplified.
Two primers, which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA region.
A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.
Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.
Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products

PCR stages

The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.
Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.
Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

Application of PCR Isolation of genomic DNA

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing . This technique may also be used to determine evolutionary relationships among organisms.

Amplification and quantitation of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques.

Variations on the basic PCR technique

Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.
Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.
Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperature|Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.
Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.
Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.
Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine,and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that deﬁned two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity. By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.
Multiplex Ligation-dependent Probe Amplification (MLPA): permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.
Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.
Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.
RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR(where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).
TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.
Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.
PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.
Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) , 5'RACE LaNe and 3'RACE LaNe .

Five steps involved in the Expression and Purification of Proteins in E. coli

2012-01-03T21:31:00.001+05:00

Production and detailed characterization of a variety of proteins is facilitated by their heterologous expression and purification. Recent advances in genomics have led to a massive increase in the number of proteins being produced using recombinant DNA technology.

In order to express heterologous proteins, a variety of expression systems have been developed. For example, bacteria (e.g. Escherichia coli, Bacillus subtilis, etc), yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, etc), filamentous fungi (e.g. Aspergillus nidulance, Trichoderma reesei, etc), insects, plant cell cultures and mammalian cell lines.

Generally, Escherichia coli is the most commonly used bacterial expression system for expression of the heterologous proteins. Reasons behind this include:

Genetic manipulation is easy,
Its culturing is inexpensive,
Expression is fast,
In many cases, the level of expression is high,
Majority of foreign proteins are well tolerated, etc.

The above mentioned advantages and many more have guaranteed that E.coli remain a valuable organism for the high-level production of heterologous proteins.

In order to carryout expression of your gene of interest in E.coli, you should follow these steps:

Choosing the Expression vector

Choosing a suitable bacterial expression vector is the first step in the expression of heterologous protein in E. coli. The choice of vector system mainly depends on two factors. These include: i) Transcriptional and translational regulators, and ii) affinity tag.

i) Transcriptional and translational regulators

These elements consist of a set of genetic components that affect both the transcription as well as translation of the heterelogous protein. These elements include:

· Promoter

Promoter is the most important transcriptional element of an expression vector. Its main function is to allow RNA polymerase to bind to the DNA. Therefore, it regulates the rate of mRNA transcription. As a result of this, the amount of heterologous protein produced, largely depends on the type of promoter used.

The promoter which results in high level of transcription is called as strong promoter. However, weak promoter is the one which allows very low level of transcription. Consequently, an expression vector should have strong promoter to carry out highest level of transcription of the cloned gene. The commonly used strong promoters in bacterial expression vectors are, lac, trp, tac, and T7 promoters.

· Repressor

Repressors are the elements which allow the repression of a promoter. When a repressor is bound to the promoter no transcription occurs. Therefore, repressor works as a regulator of promoter activity.

It is very helpful if the protein of interest is toxic to the bacteria. By the use of repressor the accumulation of toxic level of a protein is monitored. Regulation of gene expression is achieved by the calculated use of the chemical.

· Terminator

The expression vector should have both transcriptional as well as translational terminators. A transcription terminator enhances the mRNA stability, therefore, leading to substantially increased level of protein production. In addition, it also stabilizes the plasmid by checking the expression of ROP protein, which is involved in the control of copy number of the plasmid.

Translational termination of the protein is carried out by the presence of a stop codon. Bacterial expression vectors contain stop codons in all the three open reading frames, in order to prevent ribosome skipping.

· Translational initiator

Translational initiation is determined mainly by the unique structural features at the 5’ end of the mRNA. This includes the ribosome binding site (RBS). Generally, the ribosome binding site consists of Shine-Dalgarno (SD) sequence followed by an AT rich translational spacer. These elements establish the translational efficiency of the mRNA.

ii) Affinity Tag

With the advancement of gene cloning methodologies, it is now possible to construct the fusion proteins. In this strategy, specific affinity tags are added to the protein sequence of interest. Addition of affinity tag to the protein of interest, simplifies the purification of target proteins by using affinity chromatography techniques.

Apart from this, use of affinity tags has the following advantages:

· It enhances the efficiency of translation of the target mRNA.

· It protects the target protein from proteolytic degradation.

· Some of the affinity tags (e.g. maltose binding protein or MBP) help in the solubilization of the target protein, hence target proteins remain in the cytoplasm rather than inclusion bodies.

The most commonly used affinity tags are:

His-tag which bind to the immobilized metal ions (e.g. Ni²⁺).

GST-tag which binds to the glutathione–Sepharose resin.

MBP-tag which binds to the amylose resin.

Cloning the gene of interest

After choosing the suitable expression vector, the next step is to clone your gene of interest. Majority of the expression vectors provide multiple cloning sites (MCS) for the ease of cloning of the gene of interest. Generally, the MCS is placed either between the RBS and the affinity tags or between the affinity tag and the terminator. Therefore, the cloning results in the fusion of affinity tag to N or C-terminal of the target protein, respectively.

However, care should be taken in checking out the frame of the fusion protein. For example, if you are doing a N-terminal fusion, then your sequence of interest should have stop codon. Similarly, if you are doing a C-terminal fusion, then you should maintain the open reading frame with the affinity tag and your sequence of interest should have the start codon (ATG).

After doing the transformation in E.coli, it is always better to confirm cloning by sequencing. You should compare the sequences in order to find out any frame shift mutation or a miss match. These anomalies can result in the incorporation of stop codons thereby leading to premature termination of your target protein.

Expressing the recombinant protein in E.coli

After confirmation of the cloning, the next step is to transform the recombinant plasmid into the E.coli host for protein expression. You cannot use the DH5? or similar strains for expressing your protein. For the expression of heterelogous proteins, certain protease deficient strains of E. coli have been developed. The most widely used strain is the BL21 (DE3).

Transform one vial of BL21 competent cells and select them on plates having the appropriate antibiotics. It is always good to prepare a glycerol stock of a single colony. If you are checking for the first time, then you should prepare stocks of 4-5 colonies separately. It is not a good practice to store the plate at 4°C and use it in future for expression studies. Either you prepare a stock from freshly transformed E.coli or do a fresh transformation. It is observed that these strategies improve the expression of your protein of interest.

For checking the expression you can start with a culture as small as 1ml. LB is the preferred media for the expression of a wide variety of proteins. You will have to optimize the growth conditions. Different proteins are expressed in different growth conditions. These include: temperature, aeration, size of the culture, culture media, concentration of inducer, etc.

Always remember to take a culture having blank vector along with your recombinant plasmid. This will work as a negative control. If you are using an inducible system, then you should also include the un-induced sample.

After doing all these stuffs, simply run the samples on SDS-PAGE along with the protein molecular weight marker. Meanwhile calculate the molecular weight of your recombinant protein (i.e. weight of your protein of interest + weight of the affinity tag). This will help you in examining the protein gel. If you got the band of desired size in the gel, congratulations! You have done it.

Unfortunately, if you are unable to find the band of desired size, check out the above written parameters or take another colony.

It is always good to check the solubility of the recombinant protein. Confirm that the protein is soluble by loading the clear lysate along with the cell pellet. If your protein is soluble then major fraction should be visible in the clear lysate as compared to the cell pellet.

Isolating the protein

After the confirmation of expression of your protein of interest in the soluble fraction the next step is to isolate the protein. For this, the cells are harvested through centrifugation. The next step is to disrupt the cells. Various methodologies have been developed for bacterial cell disruption. The most commonly used are treatment of cells with lysozyme followed by sonication.

Generally, the harvested cells are re-suspended in the lysis buffer. The lysis buffer contains lysozyme at a concentration of around 1mg/ml. Apart from this, certain protease inhibitors like PMSF are also included. The suspension is incubated at 4°C for about half an hour. Then it is gently agitated for nearly 10 min. After this, sonication is carried out. Care should be taken while sonicating the samples. Excessive sonication leads to the denaturing of the target protein.

After disrupting the cells, the suspension is centrifuged at low rpm for around half an hour to separate the lysate from the cell debris. The supernatant obtained after centrifugation is generally called as the cleared lysate. This lysate contains your protein of interest. Now you can proceed for the purification of the protein.

Purifying the protein

After getting the clear lysate the last step is to purify the protein, exploiting the affinity tag used. Depending on the fusion tag used, a suitable resin is chosen for the purification process.

The clear lysate is first allowed to bind to the resin. This is accomplished by mixing the clear lysate along with the resin and gently agitating them for around 30 min. After this the suspension is transferred to a polypropylene column. The suspension is allowed to settle down. After this, the cap of the column is opened and lysate is allowed to pass through the resin.

The next step involves the washing of the resin with appropriate wash buffer. Care should be taken in adjusting the stringency of washing. Higher stringency will lead to the loss of the protein of interest, whereas, lower stringency will cause purification of not target proteins. Therefore, stringency should be carefully adjusted in order to get large amount of target protein with no contamination.

After washing, the protein of interest is obtained by eluting it with the appropriate elution buffer. The composition of the elution buffer depends on the fusion tag used. The isolated protein can be stored at 4°C for shorter duration (around a week) otherwise it should be stored at -20 or -80°C.

This was just the overview of steps involved in the expression and purification of proteins in E.coli. We will be publishing the detailed reviews of each step in future articles. Please feel free to ask any questions by posting it in the comments.

Modern Biotechnology 2011 (Synthetic biology) - Science of the Unthinkable.!!

2011-12-31T21:01:00.000+05:00

Three commonly used affinity tags for protein purification

2011-12-28T20:23:00.003+05:00

In order to purify heterologous proteins from various hosts, affinity tags are extremely capable tools. In the recent years affinity tags have become highly popular tools for protein purification mainly due to following reasons. They provide high level of purification of recombinant proteins from crude extracts, mostly in a single step. Second, they provide mild elution conditions, thereby, do not interfere with the structure and hence the function of the purified proteins. In addition, affinity tags allow a variety of proteins to be purified using easy procedures.

Affinity tags are available as expression vector systems having multiple cloning sites (MCS) for cloning the gene of interest towards the N or C-terminal of the tag. Now-a-days, a variety of affinity tags are available for the purification of recombinant proteins. Each tag has its certain advantages and disadvantages. Here, you will see the properties of the three commonly used affinity tags: His-Tag, GST-Tag and MBP-Tag.

1) His-Tag

His-tag is the most commonly used affinity tag for the purification of recombinant proteins in E.coli. The His-tag is a peptide motif that consists of six histidine (His) residues. Therefore, it is also called as hexa histidine-tag or 6xHis-tag. The most commonly used bacterial expression vector system for His-tag is the pET series (Novagen). These vectors provide both the N & C-terminal fusion of gene of interest with the His-tag.

It is observed that hexahistidine has high affinity towards transition metals e.g. Ni⁺⁺, Co⁺⁺ etc. Therefore, in order to purify recombinant proteins with hexa histidine-tag, immobilized-metal affinity chromatography (IMAC) columns are used. Ni-NTA agarose is the most commonly used resin for the purification of His-tag proteins. Since very few naturally occurring proteins bind to the Ni-NTA matrices with considerable affinities, therefore, recombinant proteins containing the His-Tag are significantly purified in a single step.

The recombinant protein is generally eluted either with the lowering of pH or with the addition of imidiazole to the column.

Advantages:

Hexahistidine tag is much smaller and hence provides high yields of tagged proteins.
It does not interfere with the structure and function of the recombinant protein.
Its affinity for Ni-NTA matrix is non-dependant on the conformation of the target protein.
The elution conditions for His-tagged protein are milder.
Ionic strength, chaotropic agents and detergents do not affect the purification of His-tag proteins.
Less expensive.

Because of all these advantages, His-Tag is the affinity tag of choice for various protein expression and purification experiments.

The only disadvantage of the tag is that certain bacterial proteins are able to bind to the His-Tag and hence are co-eluted with the recombinant protein. This can be partially ruled out by increasing the stringency of washing.

2) GST-Tag

The Glutathione S-transferases (GSTs) are class of enzymes that are involved in cellular defense against small toxic compounds. They are abundant enzymes that utilize glutathione as a substrate. GSTs bind to glutathione with high affinity and specificity. Therefore, they provide an efficient system for the purification of proteins.

Generally, the Glutathione S-transferase (GST) from Schistosoma japonicum is used as the affinity tag in the pGEX vector series. The gene encodes a protein of 218 residues having molecular weight of 26KDa. The fusion protein thus produced can be purified using the glutathione-based affinity resins. The strength and selectivity of the resin for GST-tagged proteins results in the successful purification of the recombinant protein from the cell extract, in a single step.

In order to elute the recombinant proteins, reduced glutathione is added to the column. This allows the elution of recombinant proteins under mild and non-denaturing conditions.

Advantages:

Provides a higher degree of purification in a single chromatographic step.
Increases the solubility of the recombinant protein.
It does not interfere with the structure and function of the recombinant protein.
Provides immunogenic as well as biochemical assay of the recombinant protein.
The elution conditions for GST-tagged protein is milder than most of the affinity purification methods.

Disadvantages:

Due to its larger size it is prone to degradation by proteases.
Affinity for glutathione resin depends on certain reagents.
More expensive.
In order to study the protein of interest in detail the tag has to be removed.

Nonetheless, GST-tag is a versatile tag for protein expression and purification. It is generally used when protein of interest is not expressed in His-tag system or is having solubility or purity issues.

3) MBP-Tag

Maltose Binding Protein (MBP) is a periplasmic protein of the bacterium E.coli. The protein is a component of the maltose and maltodextrins. It is encoded by the gene malE. The malE gene product bears a wide variety of fusions and hence is suitable for expressing proteins which do have problems in expression, in His-Tag or GST-Tag systems.

The expression vector system consisting of MBP as the affinity tag is pMAL series (New England Biolabs).

As MBP is a component of maltose, therefore, the fusion protein can be purified using matrices consisting of sugars. Generally amylose resins are used for the purification of MBP tagged proteins. For the elution of the recombinant protein maltose is added to the column. As a result of this, the elution of recombinant proteins is mild and under non-denaturing conditions.

Advantages:

Provides a higher degree of purification in a single chromatographic step.
Increases the expression as well as solubility of the recombinant protein.
It does not interfere with the structure and function of the recombinant protein.
Allows periplasmic expression of the recombinant protein.
Allows the formation of disulfide bonds in the foreign proteins.
Limits degradation of the recombinant protein.
Less expensive.

Disadvantages:

Due to its larger size, expression of large proteins is sometimes problematic .
In order to study the protein of interest in detail the MBP tag has to be removed

The elution conditions for MBP-tagged protein is milder than most of the affinity purification methods. In addition, due to bacterial origin of malE gene, MBP is a better tag in terms of expression and solubility as compared to GST.

These were the overview of the three most commonly used affinity tags for the purification of heterologous proteins in E.coli. According to our experiences, one should start with the His-Tag, followed by GST and MBP. In most of the cases, His-Tag results in decent level of expression and purification of recombinant proteins. For any query or suggestions related to protein purification in E.coli, please feel free to post it in the comments.

Kuby Immunology 4th edition by Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne

2011-12-27T13:15:00.000+05:00

Kuby Immunology is one of the top immunology textbooks on the academic market. Currently in its 6th edition, Kuby Immunology is written by a staff of top instructors in immunology. The textbook is named after Janis Kuby, a former professor at San Francisco State University and the writer of the original edition. It was originally produced as a textbook entitled Immunology, but became so well regarded in the field that it became officially designated as Kuby Immunology. Kuby Immunology is, by and large, designed as an introductory textbook, and is thus used primarily on an undergraduate level in Pre-Med and Biology programs. Kuby Immunology is highly regarded for being consistently comprehensive and up-to-date with many of the most crucial discoveries in the every changing and developing science of immunology. Since it is designed as introductory, Kuby Immunology writes out many of its concepts in straight, easy to understand terms, largely avoiding more complicated academic language. It covers most of the various categories of immunology, and focuses greatly on the multi-disciplinary nature of immunology since much of its audience does not necessarily pursue careers in immunology. For them, Kuby Immunology is the main text that they will ever read about immunology.

DOWNOAD HERE

Encyclopedia of Microbiology

2011-12-27T12:52:00.000+05:00

Humans have always struggled with how to balance the benefits bacteria offer with the threats that they produce. Much less obvious than the effects microorganisms have on plants and animals are the indirect ways in which they shape the planet. These hidden activities have rarely been explained in science, though scientists realize that the behavior of microbes supports all life on Earth.

In more than 200 entries, Encyclopedia of Microbiology presents the myriad ways in which microorganisms influence the biosphere. Focusing on how all microorganisms relate to each other just as all higher organisms relate to all other animate and inanimate objects on Earth, this new resource explores all aspects of microbiology from mycology (the study of fungi) to the simplest biological entities of all, viruses, to prions, which are even more streamlined than viruses and just as dangerous. Biographical sections in many entries highlight the scientists who most influenced developments or discoveries in microbiology, including Louis Pasteur. Entries cover new techniques in microscopy, genetic engineering, gene therapy, and nanotechnology. A full-color insert, helpful appendixes, cross-references, and further resources round out this invaluable resource.

Essays include:

Where Are Germs Found?
The Realities of Biological Weapons
Does Immigration Lead to Increased Incidence of Disease?
Will Global Warming Influence Emerging Diseases?
Microbes Meeting the Need for New Energy Sources
Bioengineered Microbes in the Environment
Does Vaccination Endanger or Improve Our Health?
Does Air Travel Make Us Sick?

DOWNLOAD HERE

5 Factors affecting gene cloning efficiency

2011-12-26T21:57:00.000+05:00

Gene cloning is the central technique involved in recombinant DNA technology. Moreover, it facilitates the discoveries and understandings of the gene structure, function and regulation. A new era has been initiated as a result of this method in the manipulation, analysis and exploitation of bio-molecules.

However, competent gene cloning is not that much easy as it sounds. In order to efficiently clone the gene of interest into a particular vector, you need to be skilled. Since the efficiency of cloning is determined by several factors. Therefore, each factor should be deliberately considered to get best cloning efficiencies.

Here we provide you the most common factors which affect the efficiency of your gene cloning experiment.

1) Starting Material

Well begun is half done. You know this notion very well. This applies to gene cloning also. A good starting material means you have done half of the things correct, only remaining half is to be optimized.

You materials should be pure. The isolated plasmid should be free from contaminating components. One of the most common contaminant is the media in which the culture is grown. This results in the poor digestion and ligation of the plasmid.

Therefore, during plasmid isolation, you should make sure that the media has been completely removed from the bacterial cells. Moreover, it is also a good practice to ethanol precipitate the plasmids prior to restriction digestion. This removes the salts present in the plasmid suspension and hence results in proper digestion of the plasmids.

If PCR product is the starting material then either reaction cleanup or gel extraction should be performed. We generally go for the gel elution of the PCR product as this not only removes the reaction components and primer dimers but also the non-specific amplifications.

2) Digestion of the Vector and Insert

Digestion is very important factor determining the efficiency of the cloning experiment. Good cloning efficiency requires complete digestion of the insert and vector molecules. Digestion, if not properly done results in partial digestion of the vector and insert molecules, which in turn results in poor cloning efficiency.

However, it is relatively uncomplicated to achieve complete digestion. You should follow the stuffs quoted here in order to get desired results.

You should take units of the restriction enzyme according to the amount of plasmid or PCR fragment taken. Generally, 1-2µg of plasmid DNA/PCR product is a good amount for digestion. To digest this amount of DNA you should take 1-2µl (5-20 units) of the chosen restriction enzyme. Now-a-days many suppliers provide enzymes in a format that contains units in 1µl to digest 1µg of DNA. This really simplifies the digestion process.

Things become more complicated when you have to perform double digestion. While performing double digestion you should always check the buffer in which both the enzymes are having maximum activity. But there are certain enzymes which cannot be used for double digestion. The reason being the fact that there buffer requirements are not compatible. In that case it is better to perform sequential digestion.

However, sequential digestion results in the loss of the fragment so you will have to start with more amount of DNA. In addition, there are certain enzymes which have different optimum temperatures. In this case also you will have to go for sequential digestion.

However, there are certain suppliers (NEB, Fermentas etc) who have optimized a single buffer and a single temperature for a variety of commonly used enzymes. Unfortunately, these enzymes are little bit expensive but in our opinion are worth. Using such enzymes not only saves your time and energy but also improves the digestion many folds.

3) Amount of digested vector to be taken for ligation

Well, after completely digesting the insert and the vector molecules, the question arises about the quantity of digested vector to be used for ligation. This significantly affects the efficiency of your gene cloning experiment.

The amount of vector to be taken solely depends on the size of the vector. For example, if the size of the vector is small then you will have to take little amount of vector and vice-versa.

We have made a generalized approach towards the size and amount of vector to be taken. According to our experiences, the optimum relation between size and amount of digested vector is as follows:

Size Amount

<5 kb 50 ng

5-7.5 kb 75 ng

7.5-10 kb 100 ng

> 10 kb upto 150 ng

Taking amount of digested vector according to its size increases the ligation to a great extent and hence the cloning efficiency.

3) Insert vs Vector Molar Ratio

Molar ratio plays a valuable role in the ligation of the fragments and hence in the cloning efficiency. But before moving further, let us see what molar ratio is? Molar ratio (sometimes also called as molar excess) is the amount of moles of insert per moles of vector molecule.

Generally, molar ratio is taken 3. That is, three insert fragments per vector molecule. It is a good practice to calculate the amount of insert in respect of vector and molar ratio prior to setting up the ligation reaction. The formula is:

ng of insert = ^{amount of vector x molar ratio x size of insert} /size of the vector

For example, if the size of your vector is 6 kb and the size of insert is 1200 bp, then for setting up the ligation reaction you should take 75 ng of vector and 45 ng of insert.

However, if the difference between the size of insert and size of vector is more (e.g. insert=300 bp; vector=12000 bp), then molar ratio should be 5-10.

In order to accurately quantify the amount of eluted insert and vector molecules you should either use spectrophotometer or a nano-drop. However, we will not recommend you to estimate the amounts by running the samples in agarose gel. This does not allow accurate quantification of the fragment and hence results in lower ligation and cloning efficiency.

5) Efficiency of the competent Cells

All well that ends well. This is also the case with gene cloning. If you have done all the things correct but you are not doing the last step properly, then you will ultimately ruin your whole hard work.

The ligation mix is used to transform E. coli competent cells and hence efficiency of the competent cells used significantly affects the cloning efficiency. We will recommend you to use high efficiency competent cells for your all cloning experiments. Always use the protocols which results in transformation efficiency of > 10⁷ cfu, for preparing competent cells.

However, if you are unable to prepare high efficiency competent cells, we will recommend you to go for commercially available competent cells. Nevertheless, you can easily prepare high efficiency competent cells, by going through our forthcoming article on competent cell preparation.

We hope that these informations will be helpful for your gene cloning experiments. However, if you have any further query regarding gene cloning, please feel free to post it in the comments.

cDNA Synthesis: Principle and Procedure

2011-12-12T22:39:00.000+05:00

With the advancement in the field of genetic engineering, gene expression analysis has become an indispensable tool. Researchers are always keen to find out whether their gene of interest is expressing (turned on) or not (turned off). For this, the mRNA (messenger RNA) is located and quantified in the given sample. mRNAs carry the information coded by DNA and, thus, further gets translated to produce respective proteins.

RNAs are very unstable and fragile, and are very likely to degrade by the omnipresent RNases. In order to combat this, the biological informations encoded in mRNA are stored in more stable form of nucleic acid, i.e. DNA. Therefore, cDNA is prepared from RNA, which stores entire sequence of the mRNA. It is more convenient to work with cDNA as compared to mRNA. This cDNA can be further used for various subsequent molecular biology and genetic studies.

What is cDNA??

cDNA means complementary DNA or copy DNA. According to the central dogma of the molecular biology, DNA is transcribed into mRNA. Then mRNA gets translated to produce protein. Therefore, the flow of biological information is from DNA to RNA to protein.

However, sometimes the flow of information is from RNA to DNA (as in the case of some viruses, e.g. HIV). This conversion of RNA to DNA is aided by an enzyme known as Reverse Transcriptase (i.e. RNA-dependent DNA polymerase). The cDNA prepared can be single stranded or double stranded. Therefore, molecular biologists make use of reverse transcriptase to prepare cDNA from mRNA for the sake of convenience in the molecular studies.

Principle of cDNA synthesis

Mature (fully spliced) mRNA is used as a template for preparing cDNA. In fact, cDNA can be produced from any RNA molecule. This conversion is brought about by reverse transcriptase. cDNA can be obtained both from prokaryotes and eukaryotes.

Reverse transcriptase is a RNA-dependent DNA polymerase. It acts on a single strand of mRNA. Using mRNA as a template, reverse transcriptase produces its complementary DNA based on the pairing of RNA base pairs. This enzyme executes reactions in the same way as DNA polymerase. It also requires a primer with a free 3′-hydroxyl group. For transcribing RNA having secondary structures, a reverse transcriptase with high temperature performance is recommended.

Procedure of cDNA synthesis

First of all, good quality intact mRNA or total RNA is isolated. Then, you need a few more reagents to prepare cDNA: dNTPs (dATP, dTTP, dCTP, dGTP), primers and reverse transcriptase.

In case of eukaryotic mRNAs, a poly-A tail is present at their 3′-ends. Therefore, a poly-T oligonucleotide is used as a primer. But certain modifications are needed when you use other RNAs which lack poly-A tail, e.g. prokaryotic mRNA, rRNA, RNA virus genomes, etc. In such cases, a poly-A tail is added to the 3′-end of the RNA. This makes it analogous to the eukaryotic mRNA.

The primer gets annealed to the 3′-end of the mRNA. Now, the 3′-end of the primer is extended with the help of the reverse transcriptase using mRNA strand as a template. This is known as “first strand reaction”. As a result of this, RNA-DNA hybrid molecule is produced. By the use of RNase H or alkaline hydrolysis, the RNA strand of this RNA-DNA hybrid molecule is digested. Now, the single stranded cDNA becomes free.

The reverse transcriptase used (most commonly used is Moloney Murine Leukemia Virus Reverse Transcriptase, MMLV RT) displays terminal transferase activity on reaching the end of the RNA template. It adds 3-5 residues (usually dC) to the 3′-terminal of the first strand cDNA. An oligo containing a stretch of G residues is used. This oligo gets annealed to the dC rich cDNA tail and serves as an extended template for reverse transcriptase. Now, the synthesis of the complementary strand of the first strand cDNA begins. This is called “second strand reaction”. Finally, a regular double stranded DNA is produced.

Types of primers used

Various types of primers can be used, in accordance to the requirements, to synthesize cDNA.

1) Oligo-dT primer- It is used when the mRNAs have poly-A tail, as in the case of eukaryotic mRNAs; or when a poly-A tail is attached to the existing RNA. Oligo-dT primer anneals to all the mRNAs simultaneously.

2) Sequence-specific primer- If you wish to generate cDNA from a particular population of mRNA among all the mRNAs, then sequence-specific primer is used. It will bind to a particular mRNA sequence only. This will give rise to a pure cDNA population generated from the desired mRNA. For designing sequence-specific primer, you must know the sequence of the mRNA of interest. Generally, the 3′-terminal sequence is preferred.

3) Random primer- A random primer cocktail is used to produce cDNA from all the mRNAs. The cDNAs produced are not full length. Random primer is extremely useful if production of the shorter cDNA fragments is desirable. Its use increases the probability of converting the entire 5′-end of the mRNA into the cDNA. In case of long mRNAs, reverse transcriptase is usually not able to reach the 5′-end. Therefore, random primer proves to be extremely advantageous in such cases.

Types of cDNA

cDNAs can be single stranded or double stranded. After the first strand reaction, cDNA obtained is single stranded. This single stranded cDNA can be converted to the double stranded form by second strand reaction. On the basis of the applications, single or double stranded form of the cDNA is used.

Applications of single stranded cDNA

1) Single stranded cDNA is most commonly used for RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). RT-PCR is done for gene expression studies. It determines whether the gene of interest is expressed or not, and the level of its expression.

2) It is also used to amplify particular gene of interest. For this, sequence-specific primers are used.

3) Real-time PCR (also known as quantitative RT-PCR, qRT-PCR) also makes use of single stranded cDNA. It is done for performing gene expression analysis. As the amplification progresses, the amplicons can be visualized with the help of a fluorescent reporter molecular. It is highly sensitive and effective as compared to RT-PCR.

Applications of double stranded cDNA

1) Double stranded cDNAs are used to clone them into the appropriate vector to prepare libraries of cDNA (i.e. cDNA libraries). These libraries contain all the mRNA sequences in the form of cDNA, which are all expressed in a cell.

2) Double stranded form of a particular cDNA of interest can be cloned. Then, expression of the desired genes is allowed at the RNA and protein level for further study.

3) Sequencing of the double stranded cDNA is carried out to obtain the expressed sequence tags (ESTs).

4) They are also used for doing microarray for analysing global gene expression.

5) Suppression subtractive hybridization (SSH) is also performed with double stranded cDNA. It is done to find out differential gene expression.

Lyophilization

2011-12-06T19:10:00.000+05:00

Biological materials often are dried to stabilize them for storage or distribution. Lyophilization also called freeze-drying, is one method of drying biological materials that minimizes damage to its internal structure

Competitive ELISA

2011-12-06T18:58:00.000+05:00

In competitive ELISA, unlabeled antibody is incubated in the presence of its antigen. Then these bound antibody/antigen complexes are then added to an antigen coated well. After washing, unbound antibodies are removed. The more analytes in the sample, the less antibodies will be able to bind to antigens in the well. The signal is then detected using labeled secondary antibodies and the decrease in signal is compared to a control. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

Indirect ELISA

2011-12-06T18:55:00.000+05:00

The indirect ELISA is used primarily to determine the strength and/or amount of antibody response in a sample. In the assay, the antigen of interested is immobilized by direct adsorption to the assay plate. Detection of the antigen can then be performed by using a matched set of primary antibody and conjugated secondary antibodies.

Sandwich ELISA

2011-12-06T18:47:00.000+05:00

The Sandwich ELISA measures the amount of analyte between capture antibody and detection antibody. The analyte needs to have two different epitope sites available for antibody binding.

Ampicillin

2011-12-04T21:20:00.000+05:00

Ampicillin is one of the most widely used antibiotic in molecular cloning, for the selection of transformants. It belongs to the penicillin group of antibiotics i.e. beta-lactam antibiotics. The only difference between penicillin and ampicillin is the presence of amino group in the latter.

Unlike penicillin which is effective against only gram positive bacteria, ampicillin is also effective against gram negative bacteria, e.g. E. coli. The amino group present in the side chain of the ampicillin renders it to penetrate the outer membrane of gram negative bacteria and hence enter into it. Therefore, it was one of the first broad spectrum penicillins to be used.

Mode of action

Like other beta-lactam antibiotics, ampicillin also inhibits cell wall synthesis in the bacteria. It basically inhibits the formation of glycan moiety of the peptidoglycan layer of the batcerial cell wall. The peptidoglycan is known to provide the rigidity to the bacterial cell wall.

Ampicillin is basically a competitive inhibitor of the peptidoglycan synthesizing enzyme transpeptidase. Inhibition of the enzyme, ultimately results in the lysis of the bacterial cells.

Ampicillin resistance gene

The gene responsible for conferring ampicillin resistance is called bla gene and codes for a TEM1 ?-lactamase enzyme. The TEM1 ?-lactamase hydrolyzes the ?-lactam ring of the ampicillin. This results in the inactivation of the antibiotic. The enzyme is usually secreted into the periplasmic space where it catalyzes the hydrolysis reaction.

TEM1 ?-lactamase is the major ?-lactamase responsible for ampicillin resistance in various gram negative bacteria including E. coli. As a result, this gene is widely used to confer resistance against ampicillin. There are a variety of vectors using bla gene to confer resistance against ampicillin for the selection of transformed E. coli.

Preparing the ampicillin stock solution

The stock solution of ampicillin is prepared in water. Generally, ampicillin sodium salt is used for its better solubility. The concentration of stock solution is generally in the range of 50-100mg/ml. We usually prepare 100mg/ml stock solutions.

Weigh 100mg of ampicillin sodium salt, dissolve it in 1ml MQ grade water and filter sterilize it by using 0.22µm filter. Store the stock at -20?C.

You should note that pH of the water has great impact on the stability of the antibiotic. It is better to use pH 6.8-7.0, of water for preparing the stock. Moreover the stock can be stored for one month only. After that the antibiotic starts to degrade.

Working concentration of ampicillin in media

For broth or agar plates the concentration of ampicillin is kept between 50-100µg/ml.

Problem of satellite colonies

As the beta lactamase is secreted in the periplasm, it degrades the ampicillin nearby the transformed cells. This results in the growth of small untransformed colonies in close proximity of the transformed one. These colonies are called as satellite colonies.

One way to deal with satellite colonies is to incubate the plates for less than 14 hours. Another way is to increase the concentration of ampicillin in the plates. We generally use 100µg/ml of ampicillin in the plates. But here also it is better to incubate the plates for less than 16 hours.

9 Factors affecting DNA extraction from agarose gel

2011-11-26T20:39:00.000+05:00

Agarose gel electrophoresis is a method of choice for the identification, purification, and separation of the DNA fragments. DNA fragments from the gel are routinely extracted for various downstream processing. These include, cloning, radio-labeling, in vitro transcription, microinjection and sequencing of the DNA molecules.

Gel extraction of DNA fragments is mainly done to remove proteins and salts that incorporate from certain reactions. Therefore, in order to use the DNA fragments for downstream processing, these components musts be removed. For example, a PCR amplification or restriction enzyme digestion reaction contains factors which inhibit further applications of the DNA fragment.

There are various methods employed for the extraction of DNA fragments from agarose gel. Among the methods used, silica-membrane containing spin-column based DNA extraction is the most widely used. This is the quickest method to effectively purify DNA fragments from agarose gels.

However, DNA extraction using this method is not always efficient and depends on following factors:

1) Size of the DNA fragment

The first factor affecting the extraction of DNA fragment from the agaorose gel is the size of the DNA fragment itself. It has been observed that DNA fragments of size 500-5000bp are extracted most efficiently from the agarose gels. However, DNA fragments smaller than 500bp or lager than 5000bp, are poorly recovered from the gel.

2) Concentration of agarose in the gel

Concentration of agarose in the gel has a significant effect on the extraction of DNA fragments. It has been observed that recovery of DNA fragments is inversely proportional to the concentration of agarose in the gel. As the concentration of agarose in the gel increases, recovery of DNA molecules decreases. Therefore, in order to carryout good recovery of DNA fragments, less concentration of agarose is used in the gel.

Generally, for gel extraction, 0.7-1% agarose gels are prepared. However, if the size of the DNA fragment is less than 300bp you will have to use higher concentration of agarose (1.2-1.5%) in the gel. We routinely use 0.7% agarose gels for extracting DNA molecules.

3) Voltage applied to the gel

The applied voltage also significantly affects the extraction of DNA fragments. In order to extract any DNA molecule from the gel you need to excise it out. For this you need a sharp and distinct band of specific DNA in the gel. Therefore, to get a sharp band, the gel has to be run using the appropriate voltage.

It has been observed that 3-5V/cm is the optimum voltage for DNA extraction. Increasing or decreasing the voltage will result in poor resolution and hence the poor recovery of the DNA fragments. For extracting the DNA fragments, we generally apply a voltage of 3V/cm to the gel.

4) Cutting of the gel slice

Cutting the gel slice also has a significant effect on the recovery of the DNA molecules. You should cut the gel slice just adjacent to the band. Moreover, extra gel portions if any should be carefully removed. It has been found that extra gel portions greatly reduce the recovery of DNA molecules.

In addition, you should try to cut small gel slices for extraction purpose. Lager gel slices incompletely solubilize and hence results in the poor recovery of the DNA molecules.

5) pH of the extraction buffer

As the adsorption of DNA fragments to silica membranes largely depends on pH, therefore, extraction of DNA fragments is significantly affected by the pH of the extraction buffer. It has been observed that maximum adsorption occurs at pH less than 7.5. As the pH of the buffer increases, adsorption of DNA to silica membrane is reduced drastically.

In order to monitor the pH of the extraction buffer, many commercially available kits add indicators to the extraction buffer. If the pH increases, color of the buffer changes, thereby enabling easy monitoring of the pH.

There are certain reasons for the increase of pH of the extraction buffer. For example, agarose gel is having incorrectly prepared high pH buffer, or the electrophoresis buffer had been repeatedly used, etc. In these cases, the pH of the extraction buffer can easily be corrected by adding a small volume of 3 M sodium acetate, pH 5.2, before proceeding.

6) Residual ethanol

After the adsorption of the DNA molecules to the silica membrane, the membrane is washed with solutions having 70% ethanol. However, before the addition of elution buffer to the membrane, the residual ethanol must be removed. It has been observed that presence of residual ethanol on the silica membrane greatly reduces the recovery of the DNA fragments.

In order to remove the residual ethanol the best way is to centrifuge the column for two minutes at maximum rpm. However, we have observed that doing so doesn’t completely remove the residual ethanol. Therefore, you should keep the column at room temperature for a while to remove the residual ethanol. But be careful, as over-drying the membrane will, in turn, result in lower recovery of the DNA fragments.

7) pH and salt concentration of the elution buffer

Elution efficiency of the DNA fragments from silica membranes strongly depends on the salt concentration and pH of the elution buffer. It has been found that most efficient elution is carried out under alkaline pH (7.5-8.5) and low salt concentrations.

Generally, the DNA fragments are eluted using 10 mM Tris-Cl, pH 8.5. However, TE buffer is not recommended for the elution of the DNA because some downstream processing may get inhibited by the presence of EDTA. Apart from 10 mM Tris-Cl, pH 8.5, MQ grade water can also be used to elute the DNA fragments. However, you will have to make sure that the pH of the water remains in the range of 7.5-8.5. Moreover, recovery of DNA fragments will be less if water is used instead of the elution buffer.

8) Volume of elution buffer

Generally, elution of DNA fragments in a spin column based extraction is carried out in small volumes. However, the volume of the elution buffer plays a significant role in the recovery of the DNA molecules. It has been observed that most efficient elution is carried out using 20-50µl of the elution buffer.

Using elution buffer less than 20µl will result in poor recovery of the DNA fragments. However, using lager volumes will result in the elution of less concentrated DNA fragments. For various purposes, we generally elute the DNA fragments in 25µl of elution buffer.

9) Incubation time of the buffer on the column

Since the main purpose of the elution buffer is to provide a medium in which the DNA fragments will be dissolved. Therefore, incubating the spin column after the addition of the elution buffer has a significant role in the recovery of the DNA molecules. Generally, it is a good practice to incubate the column for 2-5 minutes prior to centrifugation.

You can clearly see that there are a number of factors influencing the extraction of DNA fragments from agarose gel using spin column based method. Each of the above listed factors should be properly considered in order to achieve efficient extraction of the DNA.

Electricity Producing Bacteria

2011-11-26T10:08:00.000+05:00

Genetic Engineering in Livestock

2011-11-15T22:08:00.001+05:00

Recently, the first "zoofarming" product has reached market approval: it is a recombinant human protein for medical use that is produced in the milk of transgenic goats. In addition, other transgenic animals, including faster-growing salmon and „environmentally friendly" pigs with reduced levels of phosphate in their feces are awaiting regulatory approval. These are only some examples of upcoming applications of genetic engineering in farm animals. Other potential applications include traditional breeding goals such as higher milk or meat yields, leaner meat, and disease resistance. While genetic engineering in livestock opens a huge range of possibilities, it also brings about concerns of safety and justification: does genetic engineering affect animal welfare? Is it safe and morally acceptable to apply genetic engineering to farm animals for the various purposes that are envisaged?
It is against this background that the Europäische Akademie GmbH and the Berlin-Brandenburgische Akademie der Wissenschaften addressed the topic of transgenic farm animals in an interdisciplinary symposium in 2007. In these proceedings the following topics are covered: an analysis of the state of the art of the technology and its applications, an introduction to the specific application zoopharming (including its historical industrial development and the market for biopharmaceuticals), an assessment of ethical aspects, and considerations regarding the investigation of animal welfare implications of livestock biotechnology. The proceedings address science, industry, politics and the general public interested in the chances and risks of this upcoming field of biotechnology.

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Angiogenesis

2011-11-13T21:07:00.001+05:00

Angiogenesis is a normal process in growth and development, as well as in wound healing. However, this is also a fundamental step in the transition of tumors from a dormant state to a malignant state.

Sprouting angiogenesis was the first identified form of angiogenesis. It occurs in several well-characterized stages. First, biological signals known as angiogenic growth factors activate receptors present on endothelial cells present in pre-existing venular blood vessels. Second, the activated endothelial cells begin to release enzymes called proteases that degrade the basement membrane in order to allow endothelial cells to escape from the original (parent) vessel walls. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. As sprouts extend toward the source of the angiogenic stimulus, endothelial cells migrate in tandem, using adhesion molecules, the equivalent of cellular grappling hooks, called integrins. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels.

Types of angiogenesis

Sprouting angiogenesis
Intussusceptive angiogenesis

Intussusception, also known as splitting angiogenesis, was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis. First, the two opposing capillary walls establish a zone of contact. Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen. Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen. Finally, the core is fleshed out with no alterations to the basic structure. Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.

Modern Terminology of Angiogenesis

Besides the differentiation between “Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists the today more common differentiation between the following types of angiogenesis:

Vasculogenesis – Formation of vascular structures from circulating or tissue-resident endothelial stem cells (angioblasts), which proliferate into de-novo endothelial cells. This form particularly relates to the embryonal development of the vascular system.

Angiogenesis – Formation of thin-walled endothelium-lined structures with/without muscular smooth muscle wall and pericytes (fibrocytes). This form plays an important role during the adult life span, also as "repair mechanism" of damaged tissues.

Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.

Because it turned out that even this differentiation is not a sharp one, today quite often the term “Angiogenesis” is used summarizing all different types and modifications of arterial vessel growth.

Therapeutic angiogenesis

Therapeutic angiogenesis is the application of specific compounds which may inhibit or induce the creation of new blood vessels in the body in order to combat disease. The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may retard repair or some other function. Several diseases (eg. ischemic chronic wounds) are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.

The modern clinical application of the principle “angiogenesis” can be divided into two main areas: 1. Anti-angiogenic therapies (historically, research started with); 2. Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying to fight cancer and malignancies(because tumors, in general, are nutrition- and oxygen-dependent, thus being in need of adequate blood supply), the pro-angiogenic therapies are becoming more and more important in the search of new treatment options for cardiovascular diseases (the number one cause of death in the Western world). One of the world-wide first applications of usage of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease. Today, clinical research is ongoing in various clinical trials to promote therapeutic angiogenesis for a variety of atherosclerotic diseases, like coronary heart disease, peripheral arterial disease, wound healing disorders, etc.

Also, regarding the “mode of action”, pro-angiogenic methods can be differentiated into three main categories: 1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based therapies.

There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells; 2. Risk of an undesired immune response; 3 Potential toxicity, immunogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases. In contrast, pro-angiogenic protein therapy uses well defined, precisely structured proteins, with previously defined optimal doses of the individual protein for disease states, and with well-known biological effects. On the other hand, an obstacle of protein therapy is the mode of delivery: oral, intravenous, intra-arterial, or intramuscular routes of the protein’s administration are not always as effective as desired; the therapeutic protein can be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still in an early stage of research – with many open questions regarding best cell types and dosages to use.

FGF

The fibroblast growth factor (FGF) family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic FGF) consists to date of at least 22 known members. Most are 16-18 kDa single chain peptides and display high affinity to heparin and heparan sulfate. In general, FGFs stimulate a variety of cellular functions by binding to cell surface FGF-receptors in the presence of heparin proteoglycans. The FGF-receptor family is comprised of seven members and all the receptor proteins are single chain receptor tyrosine kinases that become activated through autophosphorylation induced by a mechanism of FGF mediated receptor dimerization. Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses, including cell differentiation, proliferation, and matrix dissolution – thus initiating a process of mitogenic activity critical for the growth of endothelial cells, fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members of the FGF family, can bind to all seven FGF-receptor subtypes, making it the broadest acting member of the FGF family, and a potent mitogen for the diverse cell types needed to mount an angiogenic response in damaged (hypoxic) tissues, where up regulation of FGF-receptors occurs. FGF-1 stimulates the proliferation and differentiation of all cell types necessary for building an arterial vessel, including endothelial cells and smooth muscle cells; this fact distinguishes FGF-1 from other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF) which primarily drives the formation of new capillaries.

Until now (2007), three human clinical trials have been successfully completed with FGF-1 in which the angiogenic protein was injected directly into the damaged heart muscle. Also, one additional human FGF-1 trial has been completed to promote wound healing in diabetics with chronic wounds.

Besides FGF-1, one of the most important functions of also fibroblast growth factor-2 (FGF-2 or bFGF) is the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube-like structures, thus promoting angiogenesis. FGF-2 is a more potent angiogenic factor than VEGF or PDGF (platelet-derived growth factor), however, less potent than FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are important players in wound healing. They stimulate the proliferation of fibroblasts and endothelial cells that give rise to angiogenesis and developing granulation tissue, both increase blood supply and fill up a wound space/cavity early in the wound healing process.

VEGF

VEGF (Vascular Endothelial Growth Factor) has been demonstrated to be a major contributor to angiogenesis, increasing the number of capillaries in a given network. Initial in vitro studies demonstrated that bovine capillary endothelial cells will proliferate and show signs of tube structures upon stimulation by VEGF and bFGF, although the results were more pronounced with VEGF. Upregulation of VEGF is a major component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment in vascular injuries. In vitro studies clearly demonstrate that VEGF is a potent stimulator of angiogenesis because in the presence of this growth factor plated endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries. VEGF causes a massive signaling cascade in endothelial cells. Binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producting NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. Mechanically, VEGF is upregulated with muscle contractions as a result of increased blood flow to affected areas. The increased flow also causes a large increase in the mRNA production of VEGF receptors 1 and 2. The increase in receptor production means that muscle contractions could cause upregulation of the signaling cascade relating to angiogenesis. As part of the angiogenic signaling cascade, NO is widely considered to be a major contributor to the angiogenic response because inhibition of NO significantly reduces the effects of angiogenic growth factors. However, inhibition of NO during exercise does not inhibit angiogenesis indicating that there are other factors involved in the angiogenic response.

Angiopoietins

The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies . Ang1 and Ang2 are protein growth factors which act by binding their receptors, Tie-1 and Tie-2; while this is somewhat controversial, it seems that cell signals are transmitted mostly by Tie-2; though some papers show physiologic signaling via Tie-1 as well. These receptors are tyrosine kinases. Thus, they can initiate cell signaling when ligand binding causes a dimerization that initiates phosphorylation on key tyrosines.

MMP

Another major contributor to angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries. These enzymes are highly regulated during the vessel formation process because destruction of the extracellular matrix would decrease the integrity of the microvasculature.

Applications of Angiogenesis
Tumor angiogenesis

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A tumor consists of a population of rapidly dividing and growing cancer cells. Mutations rapidly accrue within the population. These mutations (variation) allow the cancer cells (or sub-populations of cancer cells within a tumor) to develop drug resistance and escape therapy. Tumors cannot grow beyond a certain size, generally 1-2 mm³, due to a lack of oxygen and other essential nutrients.

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. Vascular Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients -- allowing for tumor expansion. On 18 July 2007 it was discovered that cancerous cells stop producing the anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG apparently limits beta-catenin which solicits angiogenesis.Other clinicians believe that angiogenesis really serves as a waste pathway, taking away the biological end products put out by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis. Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor. Evidence now suggests that the blood vessel in a given solid tumor may in fact be mosaic vessels, comprised of endothelial cells and tumor cells. This mosaicity allows for substantial shedding of tumor cells into the vasculature. The subsequent growth of such metastases will also require a supply of nutrients and oxygen or a waste disposal pathway.

Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire 'drug resistance' to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them. Recent studies by Klagsbrun, et al. have shown, however, that endothelial cells growing within tumors do carry genetic abnormalities. Thus, tumor vessels have the theoretical potential for developing acquired resistance to drugs. This is a new area of angiogenesis research being actively pursued.

Angiogenesis research is a cutting edge field in cancer research, and recent evidence also suggests that traditional therapies, such as radiation therapy, may actually work in part by targeting the genomically stable endothelial cell compartment, rather than the genomically unstable tumor cell compartment. New blood vessel formation is a relatively fragile process, subject to disruptive interference at several levels. In short, the therapy is the selection agent which is being used to kill a cell compartment. Tumor cells evolve resistance rapidly due to rapid generation time (days) and genomic instability (variation), whereas endothelial cells are a good target because of a long generation time (months) and genomic stability (low variation).

This is an example of selection in action at the cellular level, using a selection pressure to target and differentiate between varying populations of cells. The end result is the extinction of one species or population of cells (endothelial cells), followed by the collapse of the ecosystem (the tumor) due either to nutrient deprivation or self-pollution from the destruction of necessary waste pathways.

Angiogenesis-based tumour therapy relies on natural and synthetic angiogenesis inhibitors like angiostatin, endostatin and tumstatin. These are proteins that mainly originate as specific fragments pre-existing structural proteins like collagen or plasminogen.

Recently, the 1st FDA-approved therapy targeted at angiogenesis in cancer came on the market in the US. This is a monoclonal antibody directed against an isoform of VEGF. The commercial name of this antibody is Avastin, and the therapy has been approved for use in colorectal cancer in combination with established chemotherapy.

Angiogenesis for cardiovascular disease

Angiogenesis represents an excellent therapeutic target for the treatment of cardiovascular disease. It is a potent, physiological process that underlies the natural manner in which our bodies respond to a diminution of blood supply to vital organs, namely the production of new collateral vessels to overcome the ischemic insult. Perhaps the greatest reason for these trials’ failure to achieve success is the high occurrence of the “placebo effect” in studies employing treadmill exercise test readout. Thus, even though a majority of the treated patients in these trials experience relief of such clinical symptoms such as chest pain (angina), and generally performed better on most efficacy readouts, there were enough “responders” in the blinded placebo groups to render the trial inconclusive. In addition to the placebo effect, more recent animal studies have also highlighted various factors that may inhibit an angiogenesis response including certain drugs, smoking, and hypercholesterolemia.

Although shown to be relatively safe therapies, not one angiogenic therapeutic has yet made it through the gauntlet of clinical testing required for drug approval. By capitalizing on the large database of what did and did not work in previous clinical trials, results from more recent studies with redesigned clinical protocols give renewed hope that angiogenesis therapy will be a treatment choice for sufferers of cardiovascular disease resulting from occluded and/or stenotic vessels.

Early clinical studies with protein-based therapeutics largely focused on the intravenous or intracoronary administration of a particular growth factor to stimulate angiogenesis in the affected tissue or organ. Most of these trials did not achieve statistically significant improvements in their clinical endpoints. This ultimately led to an abandonment of this approach and a widespread belief in the field that protein therapy, especially with a single agent, was not a viable option to treat ischemic cardiovascular disease. However, the failure of gene- or cell-based therapy to deliver, as of yet, a suitable treatment choice for diseases resulting from poor blood flow, has led to a resurgence of interest in returning to protein-based therapy to stimulate angiogenesis. Lessons learned from earlier protein-based studies, which indicated that intravenous or intracoronary delivery of the protein was not efficacious, have led to completed and ongoing clinical trials in which the angiogenic protein is injected directly into the beating ischemic heart.

Such localized administration of the potent angiogenic growth factor, human FGF-1, has recently given promising results in clinical trials in no-option heart patients. Angiogenesis was documented by angiographically visible “blushing”, and functional exercise tests were also performed on a subset of patients. The attractiveness of protein therapy is that large amounts of the therapeutic agent can be injected into the ischemic area of interest, to pharmacologically start the process of blood vessel growth and collateral arteries’ formation. In addition, from pharmacokinetic data collected from the recent FGF-1 studies in the human heart, it appears that FGF-1, once it exits the heart is cleared in less than three hours from the circulation. This would presumably prevent FGF-1 from stimulating unwanted angiogenesis in other tissues of the bodies where it could potentially cause harm, such as the retina and in the kidneys. No serious adverse events have yet to be noted in any of the completed or ongoing clinical trials in which the FGF-1 protein is utilized as the therapeutic agent tom stimulate angiogenesis

Production of biodiesel from Bacteria

2011-09-27T22:07:00.001+05:00

How to Prevent Dengue?

2011-09-26T14:24:00.002+05:00

What is dengue?

Dengue fever is an acute viral disease caused by the Flavivirus of the family Flaviviridae. The term “dengue” is a Spanish attempt at the Swahili phrase “Ki denga pepo” meaning “cramp-like seizure” caused by an evil spirit.

It’s a disease of tropical and subtropical regions that occurs epidemically, very much similar to chikungunya.

This disease is also called “breakbone” fever because it sometimes causes severe joint & muscle pain that feels like bones are breaking, hence the name.

The illness is usually self-limiting and can last up to 10 days, but complete recovery can take as long as a month.

How dengue spreads?

Dengue fever is noncontagious i.e., not an airborne infection (an infected person cannot spread the infection to other persons but can be a source of dengue virus for mosquitoes for about 6 days from the start of symptoms).

Dengue virus is transmitted to humans through mosquito bites, a specific species of mosquito usually Aedes aegypti (but frequently Aedes albopictus) which bites during morning hours. The mosquito transmits disease by biting an infected person and then biting someone else, similar to the spread of chikungunya.

The incubation period (i.e., period from infection till the manifestation of symptoms) is 4 to 6 days, but may vary with a range of 3 to 14 days.

This disease is a vector borne infection i.e., mosquito is the vector (carrier) of the virus believed to cause this fever and the vector is common both in dengue and chikungunya.

Dengue symptoms

Symptoms of typical (classic) dengue usually start with fever within 5 to 6 days after someone is bitten by an infected mosquito. Symptoms are more or less similar to that ofchikungunya and include:

High-grade fever.
Severe headache.
Severe joint and muscle pain.
Nausea and vomiting.
Skin rash – The rash may appear over most of the body 3 to 4 days after fever.
Bleeding from the nose, gums or under the skin, causing purplish bruises.

Dengue severity classification

Four grades of severity are recognized: Grade I, fever and constitutional symptoms; grade II, grade I plus spontaneous bleeding (of skin, gums, or gastrointestinal tract); grade III, grade II plus agitation and circulatory failure; grade IV, profound shock.

Dengue synonyms

Exanthesis arthrosia, Aden fever, bouquet fever, breakbone fever, dandy fever, date fever, dengue fever, dengue hemorrhagic fever, polka fever, solar fever, scarlatina rheumatica.

Dengue diagnosis

Dengue fever can be diagnosed by performing blood test to detect antibodies against the virus.

How to prevent dengue?

Prevention is basically by:

Avoiding mosquito bites (by using mosquito repellents containing DEET, picaridin(KBR3023) or oil of lemon eucalyptus)
Eliminating pockets of stagnant water that serve as mosquito breeding sites at home, workplaces and their vicinity,
Not storing water in open containers. Covering all water containers with lids.
Preventing mosquito entry by keeping doors closed and windows screened.
Wearing protective clothing like long-sleeved shirts, long pants, socks and shoes when outdoors.
Using mosquito nets at home.
Scrubbing and cleaning margins of containers used for water (to dislodge the eggs of Aedes aegypti)
Covering overhead tank to prevent access to mosquitoes.
Aedes mosquitoes usually bite during the day; therefore, special precautions should be taken during early morning hours before day break and in the late afternoon before dark.
There is no commercially available dengue vaccine (for dengue Flavivirus).

Dengue treatment and prognosis

Prognosis for dengue fever is good, if the infectious disease is treated on time. However the mortality rate can be as high as 15%. Hence immediate medical attention should be sought in suspected cases with dengue fever.

Scientists edge nearer unlimited blood bank

2011-09-25T14:24:00.000+05:00

French scientists have managed to generate red blood cells from stem cells and inject them back in to the donor. This major achievement opens up the possibility of a stem cell-based alternative to donated blood cells.

Waste Water + Bacteria = Clean Energy

2011-09-23T12:12:00.000+05:00

For the first time, researchers have sustainably produced hydrogen gas, a potential source of clean energy, using only water and bacteria. The challenge now, scientists say, is to scale up the process to provide large amounts of hydrogen for various purposes, such as fueling vehicles or small generators.

Hydrogen may be the ultimate clean fuel because burning it—in chemical terms, reacting it with oxygen—yields only water vapor. Previously, researchers have produced hydrogen gas in microbial-powered, batterylike fuel cells, but only when they supplemented the energy produced by the bacteria with electrical energy from external sources—such as that obtained from renewable sources or burning fossil fuels, says Bruce Logan, an environmental engineer at Pennsylvania State University, University Park. Also, by using devices that contain large stretches of permeable membranes that separate salt water from fresh, scientists have tapped the voltage difference that exists between them. But those devices create only a voltage difference; they don't generate the electrical current required to produce hydrogen, Logan notes. Hydrogen atoms are formed in such devices only when electrons flow into a fluid where they can combine with hydrogen ions; those atoms in turn combine with each other to create hydrogen gas.

Bacterial gas. Using a prototype system that uses only fresh water (bottle, left), salt water (right), and a chamber where certain types of energy-generating bacteria feed on nutrients (foreground), scientists have produced hydrogen gas (collected in chamber at arrow) without using any external sources of energy.

Now, Logan and Penn State environmental engineer Younggy Kim report online this week in the Proceedings of the National Academy of Sciences that they've done something no other team has: They've successfully combined the two types of devices to generate hydrogen without any external sources of energy whatsoever. The prototype device contains two small chambers—one holding the bacteria and their nutrients, the other holding salty water where the hydrogen was produced—that are separated by five stacked cells through which the researchers circulated fresh water and salt water. Together, these stacked cells generated between 0.5 and 0.6 volts—enough, the researchers say, to enable hydrogen production in the microbial fuel cell, in which bacteria feed on acetate compounds.

For each 30 milliliters of sodium acetate solution provided for the bacteria, the device generated between 21 and 26 milliliters of hydrogen gas over the course of a day. Admittedly, this is a small volume, about four times the amount of fuel in a disposable lighter, but it's enough to prove that the hydrogen-generating concept works in the lab, the researchers contend. Although the equipment needed to produce the hydrogen is expensive, the device needs no external source of energy—and therefore no greenhouse gases are generated during the process.

The team's device "is elegantly simple, and their test results are well-explained and unambiguous," says Leonard Tender, a chemist at the U.S. Naval Research Laboratory in Washington, D.C. One of the challenges to scaling up the process, he notes, will be developing new materials for fuel cell membranes that won't quickly become clogged with the chemical byproducts of bacterial activity, which would cut down on the flow of ions that help maintain the voltage difference across the membranes. Once such hurdles are crossed, however, the process offers the intriguing possibility of using the organic matter in wastewater to generate energy, he notes.

But César Torres, a chemical engineer at Arizona State University, Tempe, suggests that the new technology isn't quite ready for full-scale production of hydrogen. "This is a simple process, but the chemistry and the components are complicated," he says. "The technology needed to design and manufacture materials needed to produce efficient, nonclogging membranes is quickly evolving, but there's still a lot of research to be done."

Another challenge to scaling up will be "keeping the bacteria happy," he notes. The key, he suggests, will be extracting much but not all the energy produced by the bacteria. Trying to use all of the energy produced by bacterial metabolism wouldn't leave enough for the microbes to grow, reproduce, and thrive.

HIV-Mode of action of NNRTIs

2011-08-16T22:57:00.000+05:00

NNRTIs are a class of anti-HIV drugs. When one NNRTI is used in combination with other anti-HIV drugs – usually a total of 3 drugs – then this combination therapy can block the replication of HIV in a person's blood.

NNRTIs, sometimes referred to as "Non-Nucleoside Analogues" – or "non-nukes" for short – prevent healthy T-cells in the body from becoming infected with HIV.

When HIV infects a cell in a person's body, it copies it's own genetic code into the cell's DNA. In this way, the cell is then "programmed" to create new copies of HIV. HIV's genetic material is in the form of RNA. In order for it to infect T-cells, it must first convert its RNA into DNA. HIV's reverse transcriptase enzyme is needed to perform this process.

NNRTIs attach themselves to reverse transcriptase and prevent the enzyme from converting RNA to DNA. In turn, HIV's genetic material cannot be incorporated into the healthy genetic material of the cell, and prevents the cell from producing new virus.

DNA Helicase: Function, Structural Features And Superfamilies

2011-07-20T20:28:00.000+05:00

DNA helicase is an enzyme that aids in DNA synthesis by 'unzipping' the two strands of a DNA helix so that DNA polymerase can access the DNA to add nucleotides and effect copying.

Many cellular processes (DNA replication, RNA transcription, DNA recombination, DNA repair, Ribosome biogenesis) involve the separation of nucleic acid strands. Helicases are often utilized to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP or GTP hydrolysis. They move incrementally along one nucleic acid strand of the duplex with a directionality specific to each particular enzyme. There are many helicases (14 confirmed in E. coli, 24 in human cells) resulting from the great variety of processes in which strand separation must be catalyzed.

Function

Helicases adopt different structures and oligomerization states. Whereas DnaB-like helicases unwind DNA as donut shaped hexamers, other enzymes have been shown to be active as monomers or dimers. Recent studies showed that helicases do not merely wait passively for the fork to widen, but play an active role in forcing the fork to open, thus "it is an active unwinding motor". However, the unwinding is much faster in cells than in the test tube, so "accessory proteins are helping the helicase out by destabilizing the fork junction".

Structural features

The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology; they all possess common sequence motifs located in the interior of their primary sequence. These are thought to be specifically involved in ATP binding, ATP hydrolysis and translocation on the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.

Based on the presence of defined helicase motifs, it is possible to attribute a putative helicase activity to a given protein, though the presence of a motif does not confirm the protein as a helicase. Conserved motifs do, however, support an evolutionary homology among enzymes. Based on the presence and the form of helicase motifs, helicases have been separated in 4 superfamilies and 2 smaller families. Some members of these families are indicated, with the organism from which they are extracted, and their function.

Superfamilies

Superfamily I: UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA (Staphylococcus aureus, Bacillus anthracis and Bacillus cereus, regulation of recombination by displacing RecA from DNA and inhibiting RecA-mediated DNA strand exchange), Dda (bacteriophage T4, replication initiation).
Superfamily II: RecQ (E. coli, DNA repair), eIF4A (Baker's Yeast, RNA translation), WRN (human, DNA repair), NS3 (Hepatitis C virus, replication). TRCF (Mfd) (E.coli, transcription-repair coupling factor).
Superfamily III: LTag (Simian Virus 40, replication), E1 (human papillomavirus, replication), Rep (Adeno-Associated Virus, replication, site-specific integration, virion packaging).
DnaB-like family: DnaB (E. coli, replication), gp41 (bacteriophage T4, DNA replication),T7gp4 (bacteriophage T7, DNA replication).
Rho-like family: Rho (E. coli, Transcription termination factor ).

Lecture on Stem Cells and the End of Aging

2011-07-14T19:18:00.000+05:00

Human tissues vary in their ability to heal and regenerate. The nervous system has weak powers of regeneration, while the skin is quick to make new cells for repair. Mammalian muscle cells are intermediate in their ability to regenerate. Human muscle can regenerate in response to minor wounds and normal wear and tear, but humans will not grow a new bicep, for example, in response to amputation. The heart is the most important muscle in the body and yet has feeble regenerative capabilities. Research into the wholesale production of new replacement organs and limbs is in its infancy, but research into enhancing normal levels of regeneration is progressing rapidly. Recent discoveries concerning the location and characteristics of adult stem cells and the signals that wounded tissue produces to activate stem cells have increased our understanding of regeneration. Insulin-like growth factor 1 (IGF1) is an example of an important stem cell communication molecule. If the activity of the growth factor is experimentally enhanced, muscle regeneration improves.

DNA Repair

2011-06-29T20:32:00.000+05:00

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day.Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. Consequently, the DNA repair process is constantly active as it responds to damage in the DNA structure.

The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:

1. an irreversible state of dormancy, known as senescence
2. cell suicide, also known as apoptosis or programmed cell death
3. unregulated cell division, which can lead to the formation of a tumor that is cancerous

The DNA repair ability of a cell is vital to the integrity of its genome and thus to its normal functioning and that of the organism. Many genes that were initially shown to influence lifespan have turned out to be involved in DNA damage repair and protection. Failure to correct molecular lesions in cells that form gametes can introduce mutations into the genomes of the offspring and thus influence the rate of evolution.

DNA damage

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

DNA repair mechanisms

Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when so-called "non-essential" genes are missing or damaged). Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to losslessly recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort.

Damage to DNA alters the spatial configuration of the helix and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place. The types of molecules involved and the mechanism of repair that is mobilized depend on the type of damage that has occurred and the phase of the cell cycle that the cell is in.

Direct reversal

Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can only occur in one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of thymine dimers (a common type of cyclobutyl dimer) upon irradiation with UV light results in an abnormal covalent bond between adjacent thymidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300–500nm wavelength) to promote catalysis. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called as ogt. This is an expensive process because each MGMT molecule can only be used once; that is, the reaction is stoichiometric rather than catalytic.A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes. The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.

Base excision repair (BER), which repairs damage to a single nucleotide caused by oxidation, alkylation, hydrolysis, or deamination. The base is removed with glycosylase and ultimately replaced by repair synthesis with DNA ligase.
Nucleotide excision repair (NER), which repairs damage affecting longer strands of 2–30 bases. This process recognizes bulky, helix-distorting changes such as thymine dimers as well as single-strand breaks (repaired with enzymes such UvrABC endonuclease). A specialized form of NER known as Transcription-Coupled Repair (TCR) deploys high-priority NER repair enzymes to genes that are being actively transcribed.
Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but normal, that is non- damaged) nucleotides following DNA replication.

Double-strand breaks

Double-strand breaks (DSBs), in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. Two mechanisms exist to repair DSBs: non-homologous end joining (NHEJ) and recombinational repair

, a specialized DNA Ligase that forms a complex with the cofactor XRCC4, directly joins the two ends. To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate. NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are "backup" NHEJ pathways in higher eukaryotes. Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system.

Recombinational repair requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.

Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.

A team of French researchers bombarded Deinococcus radiodurans to study the mechanism of double-strand break DNA repair in that organism. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination.

Translesion synthesis

Translesion synthesis is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites. It involves the switching out of regular DNA polymerases for specialized translesion polymerases, often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol η mediates error-free bypass of lesions induced by UV irradiation, whereas Pol ζ introduces mutations at these sites. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death.

Taq Polymerase Enzyme

2011-06-28T23:32:00.000+05:00

Taq polymerase is a thermostable DNA-dependent DNA polymerase that catalyzes the template-directed polymerization of dNTPs at high temperatures. Taq Polymerase was first isolated in 1976 from Thermus aquaticus strain YT-1.

The Properties of Taq Polymerase Enzyme

Taq Polymerase catalyzes the DNA-dependent polymerization of dNTPs, one unit of the enzyme is defined as the amount of enzyme that will incorporate 10 nmol of radioactively labeled dTTP into acid insoluble material at 80^oC in 30 minutes. The enzyme could be purified to a specific activity of 200000 U/mg.
The activity of the enzyme is dependent on bivalent cations, such as Mg²⁺. The optimum concentration of₂ is 2 mM. Monovalent cations also have an effect on the activity of Taq Polymerase. The monovalent cation is K⁺ in a form of KCl when it is using with the optimum concentration 50 mM, when KCl concentration more than 75 mM it can inhibit the activity of taq polymerase. Another monovalent cations, such as NaCl, NH₄Cl and NH₄acetate, cannot substitute KCl without a decrease in specific activity. MgCl
Maximum polymerization rates of Taq Polymerase are obtained with 0.7-0.8 mM dNTPs. While at dNTP concentrations of 4-6 mM, substrate inhibition is observed. Denaturing agents, detergents, and organic solvents in low concentration are tolerated by Taq Polymerase, while at higher concentrations, the inhibition of enzyme activity is observed.
The major distinguishing feature of Taq Polymerase is its extreme thermal stability. The enzyme can withstand temperatures in excess of 95^oC for prolonged periods, and in fact, its optimum for reaction is 75^oC. The rate of reaction is reduced to 50% at 60°C, and to 10% at 37^oC.

Taq Polymerase and PCR Technique

The major application of Taq polymerase at present is in the polymerase chain reaction (PCR). This technique is a simple method of amplifying minute quantities of DNA for a variety of subsequent procedures, including cloning, sequencing, hybridization, and genome mapping. The application of taq polymerase to the PCR was the basis for the success of the technique. It is caused by these following factors:

Taq polymerase enzyme is stable up to 95^oC, so it is not necessary to replenish the enzyme after each PCR cycle.
The activity of the enzyme is maximum at the temperature between 70 and 75^oC, which minimizes secondary structures of the template, resulting in high polymerization yield.
The annealing can be chosen from 30-70^oC, allowing an optimal adaptation of cycle parameters to appropriate annealing temperatures of the primers; therefore, by products are hardly generated.

The enzyme from Thermus thermophilus (when used in a manganese-containing buffer) has an additional reverse transcriptase activity, which extends the use of PCR directly to cDNA synthesis. Another use of Taq polymerase 1s directly in DNA sequencing, where the high temperatures employed help reduce problems caused by secondary structure in the template and allow an increase in the stringency of primers used.

Protein Hydrolysis: Acid And Alkaline Method

2011-06-24T23:23:00.000+05:00

There are three general methods to hydrolyze protein into its composition, amino acids. Those methods are acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Strong acid is ordinarily the method of choice, and constant boiling hydrochloric acid, 6 M, is most frequently used. The reaction is usually carried out in evacuated sealed tubes or under N2 (Nitrogen) at110 Celcius degree for 18 to 96 hours. Under these conditions, peptide bonds are quantitatively hydrolyzed (although relatively long periods are required for the complete hydrolysis of bonds to valine, leucine, and isoleucine).

While the complete alkaline hydrolysis of proteins, is achieved with 2 to 4 M sodium hydroxide at 100 Celcius degree for 4 to 8 hours. This is of limited application for routine analysis, because cysteine, serine, threonine, and arginine are destroyed in the process, and partial destruction by deamination of other amino acids occurs. The complete enzymatic hydrolysis of proteins is difficult, because most enzymes attack only specific peptide bonds rapidly.

In this particular I only provide two methods of protein hydrolysis, acid hydrolysis and alkaline hydrolysis. Here are the methods:

Materials that you need:

3M p-toluenesulfonic acid.

0.2% tryptamine 3-[2-Aminoethyl] indole.

3M mercaptoethanesulfonic acid (Pierce).

1M sodium hydroxide.

Acid Hydrolysis of Protein:

1 mL of 3M p-toluenesulphonic acid, containing 0.2% tryptamine (0.2% 3-[2-aminoethyl] indole) is added to the protein dried in a Pyrex glass tube (1.2 x 6 cm or similar, in which a constriction has been made by heating in an oxygen/gas flame).
The solution is sealed under vacuum and heated in an oven for 24 to 72 hours at 110 Celcius degree.
Altematively, you can use 3M mercaptoethanesulfonic acid as p-toluenesulphonic acid replacing, The sample is hydrolyzed for a similar time and temperature.
The tube is allowed to cool and cracked open with a heated glass rod held against a horizontal scratch made in the side of the tube.
The acid is taken to near neutrality by carefully adding 2 mililiters of 1M sodium hydroxide.
After this hydrolysis you can continue carrying out to quantitatively analyze certain amino acids, such as tryptophan.

Alkaline Hydrolysis Protein:

0.5 mL of 3M sodium hydroxide is added to the protein dried in a Pyrex glass tube.
The solution is sealed under vacuum and heated in an oven for 4 to 8 hours at 100 Celcius degree.
After cooling and cracking open, the alkali is neutralized carefully with an equivalent amount of 1M HCl.

Epigenome

2011-06-21T20:43:00.000+05:00

The "epigenome" is a parallel to the word "genome", and refers to the overall epigenetic state of a cell. The phrase "genetic code" has also been adapted—the "epigenetic code" has been used to describe the set of epigenetic features that create different phenotypes in different cells. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an epigenomic map, a diagrammatic representation of the gene expression, DNA methylation and histone modification status of a particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the histone code or DNA methylation patterns.

Quantitative Estimation of DNA Concentrations

2011-05-27T23:01:00.000+05:00

DNA, RNA, and protein strongly absorb ultraviolet light in the 260 to 280 nm range. UV spectroscopy can be used as a quantitative technique to measure nucleic acid concentration and protein contamination. Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm while proteins do the opposite. The general rules for determining the concentrations of nucleic acids at 260 nm are:

1 Optical Density (OD) unit of double-stranded DNA is 50 micrograms/ml.
1 OD unit of single-stranded DNA is 33 micrograms/ml.
1 OD unit of single-stranded RNA is 40 micrograms/ml.

Proteins absorb strongly at 280 nm where 1 OD unit is 1 mg/ml. When using UV spectroscopy for estimating DNA concentrations, it is very important to remove all protein and RNA from the DNA solution. Good estimations can only be made on clean preparations.

An estimate of the purity of a DNA preparation can be made by measuring the absorbance at both 260 nm and 280 nm. Pure solutions of nucleic acid will absorb approximately twice as much at 260 nm as at 280 nm. Experimentally, the ratio of 260 nm/280 nm of a pure DNA solution is between 1.8 to 2.0. As protein contamination increases, the ratio decreases. Additionally, the presence of contaminating oranic solvents, such as phenol, can affect estimations of concentration and purity.

Materials you need are:

UV Spectrophotometer

Quartz or UV compatible cuvettes

TE buffer

DNA template

Method:

Fill the cuvette with water or TE buffer. Zero the spectrophotometer at 260 nm with this blank.
DNA from plasmid and genomic preparations is typically at a concentration exceeding 1 micrograms/microliter. Consequently, DNA is usually diluted before measuring its absorbance. An unfortunate result of this measurement is that the DNA is expended as a result of the dilution. Be sure these is adequate DNA to waste. Start by diluting the DNA sample 1 microliter : 999 microliters of TE buffer (the dilution can be done directly in the cuvette). Mix the dilution thoroughly.
Measure the optical density (OD). Multiply the resulting OD by 50 micrograms/ml. For a 1:1000 dilution, the mass of DNA is equal to micrograms/microliter.
Similarly, the same sample can be measured at 280 nm. A ratio of the OD-260nm/OD-280nm is an indicator of DNA purity. A ratio of 1.8 or higher indicates minimal protein contamination.

Isolation Of Genomic DNA Rrom Yeast

2011-05-27T22:46:00.002+05:00

Isolating genomic DNA from yeast involves culturing the microbe, harvesting the cell, enzymatically removing the cell wall, lysing the protoplast, and finally separating the DNA from the other cell debris.
Materials that you need are:

Yeast culture-prepared previously

Spectrophotometer with cuvettes

50 mM EDTA, pH 8-ice cold

50 mM Tris, pH 9.5, 2% 2-mercaptoethanol

1.2 M sorbitol, 50 mM Tris, pH 7.5

Lyticase solution-500 U/ml in 50 mM Tris, pH 7.5

10% Sodium Dodecyl Sulfate (SDS)-used for checking protoplast formation

Lysis buffer-100 mM Tris, pH 7.5, 100 mM EDTA, 150 mMNaCl, 50 micrograms/ml RNase A

Lysis buffer with 2% SDS

95% Ethanol-stored at minus 20 degree Celcius

TE buffer-10 M Tris, pH 8, 1 mM EDTA

3 M potassium acetate, pH 5.5

Here are the step by step methods:

The yeast can be cultured for as long as 48 hours at 30 degree Celcius. The optical density of a 1:10 dilution of the culture in water can be as high as 1.0 at 520 nm.
Harvest 5 ml of cells by centrifugation (5 minutes at 5000 rpm). Resuspend the yeast in 1 ml of cold 50 mM EDTA, pH 8, and transfer to a 1.5 ml microfuge tube. Centrifuge for 1 minute, decant, and resuspend again in 50 mM EDTA.
Pellet the cells as before and suspend the cells in 1 ml of 50 mM Tris, pH 9.5, 2% 2-mercaptoethanol. Incubate for 10 min at room temperature. Centrifuge and decant.
Resuspend the cells in 800 micro liter of 1.2 M sorbitol, 50 mM Tris, pH 7.5. The sorbitol act as an osmotic support and prevents rupture of the cells as the wall is removed. As the yeast cell walls degrade, membranes can easily overextend and rupture.
Add 200 micro liter of Lyticase (500 U/ml in 50 mM Tris, pH 7.5). Place the cells on a rocker and incubate at 37 degree Celcius for one hour. Lyticase is a yeast cell wall degrading enzyme isolated from the bacteria Arthrobacter luteus.
Examine the suspension under a microscope to ensure protoplast formation. As the yeast wall is degraded, the cell membrane can ooze out of the sack. Viewed with phase contrast microscopy, yeast protoplasts are characteristically refractile (or bright) spheres, and yeast cell wall shells appear as gray ghosts (cell walls without membrane and cytosol). Combine 10 micro liter of 10% SDS with 10 micro liter of yeast protoplasts. Examine the cells under the microscope. The absence of refractile yeast indicates the protoplasts were lysed by the SDS.
Pellet the protoplasts by centrifuging at 10000 rpm for five minutes. Resuspend the cells in 1 ml of 100 mM Tris, pH 7.5, 100 mM EDTA, 150 mM NaCl (lysis buffer). Transfer the cells to a 5 ml polypropylene tube. Add 1 ml of lysis buffer with 2% SDS. Mix and incubate at 30 Celcius egree for 30 minutes. Check the cells under a microscope for lysis.
Centrifuge the lysate at 5000 rpm for 15 min to pellet cellular debris. Decant the upper phase containing the DNA.
Using a pipet, determine the volume of the DNA solution. Add 1/10th volume (e.g., 100 micro liter for every ml) of 3 M potassium acetate to the solution. In the presence of ions Na and K, DNA precipitates if mixed with either ethanol or isopropanol. Incubate the DNA at -20 Celcius degree for 30 min (or overnight if possible. Centrifuge the solution at 7000 rpm for 20 minutes. The DNA appears as white pellet. Decant and remove as much moisture as possible, but do not allow the pellet to dry. Once genomic DNA drys, it can be very difficult to resuspend.
Resuspend the DNA in 100 micro liter of TE buffer and freeze.

Gold Nanoparticles In Cancer Cell Detection

2011-05-06T23:05:00.000+05:00

“Gold nanoparticles are very good at scattering and absorbing light,” said Mostafa El-Sayed, director of the Laser Dyanamics Laboratory and chemistry professor at Georgia Tech. “We wanted to see if we could harness that scattering property in a living cell to make cancer detection easier. So far, the results are extremely promising.”

Many cancer cells have a protein, known as Epidermal Growth Factor Receptor (EFGR), all over their surface, while healthy cells typically do not express the protein as strongly. By conjugating, or binding, the gold nanoparticles to an antibody for EFGR, suitably named anti-EFGR, researchers were able to get the nanoparticles to attach themselves to the cancer cells.

“If you add this conjugated nanoparticle solution to healthy cells and cancerous cells and you look at the image, you can tell with a simple microscope that the whole cancer cell is shining,” said El-Sayed. “The healthy cell doesn’t bind to the nanoparticles specifically, so you don’t see where the cells are. With this technique, if you see a well defined cell glowing, that’s cancer.”

In the study, researchers found that the gold nanoparticles have 600 percent greater affinity for cancer cells than for noncancerous cells. The particles that worked the best were 35 nanometers in size. Researchers tested their technique using cell cultures of two different types of oral cancer and one nonmalignant cell line. The shape of the strong absorption spectrum of the gold nanoparticles are also found to distinguish between cancer cells and noncancerous cells.

What makes this technique so promising, said El-Sayed, is that it doesn’t require expensive high-powered microscopes or lasers to view the results, as other techniques require. All it takes is a simple, inexpensive microscope and white light.

Another benefit is that the results are instantaneous. “If you take cells from a cancer stricken tissue and spray them with these gold nanoparticles that have this antibody you can see the results immediately. The scattering is so strong that you can detect a single particle,” said El-Sayed.

Finally, the technique isn’t toxic to human cells. A similar technique using artificial atoms known as Quantum Dots uses semiconductor crystals to mark cancer cells, but the semiconductor material is potentially toxic to the cells and humans.

Human Development and Stem Cells

2011-04-15T20:35:00.000+05:00

Human embryonic development depends on stem cells. During the course of development, cells divide, migrate, and specialize. Early in development, a group of cells called the inner cell mass (ICM) forms. These cells are able to produce all the tissues of the body. Later in development, during gastrulation, the three germ layers form, and most cells become more restricted in the types of cells that they can produce.

Real-time PCR in Microbiology: From Diagnosis to Characterization

2011-04-12T11:51:00.000+05:00

Ian M. MacKay "Real-time PCR in Microbiology: From Diagnosis to Characterization"

Publisher: Caister Academic Press 2007 | 454 Pages | ISBN: 1904455182 | PDF | 15.8 MB
Description or mention of instrumentation, sofhvare, or other products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of
their use. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the publisher. No claim to original U.S. Government works.

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Scripps Research Scientists Find E. Coli Enzyme Must Move to Function

2011-04-12T11:20:00.000+05:00

Scripps Research Scientists Find E. Coli Enzyme Must Move to Function

Slight oscillations lasting just milliseconds have a huge impact on an enzyme's function, according to a new study by Scripps Research Institute scientists. Blocking these movements, without changing the enzyme's overall structure or any of its other properties, renders the enzyme defective in carrying out chemical reactions.

The study, published in the April 8, 2011 issue of the journal Science, adds to a growing body of evidence pointing to the importance of movement in the ability of enzymes and other types of proteins to do their job. The findings may also help scientists design more specific and effective drugs targeting enzymes.

"Ever since the first X-ray structures of proteins emerged, scientists have been talking about proteins as though their structures were fixed in space," said Peter Wright, chair of the Department of Molecular Biology and member of the Skaggs Institute for Chemical Biology at Scripps Research, who was senior author of the study. "But that is not how proteins work. They are like the machines we build. They have moving parts, and they need motion to work."

A Model Enzyme

The new study examined the enzyme dihydrofolate reductase (DHFR) from the common bacterium Escherichia coli, which the Wright group has been using as a model for understanding how enzymes catalyze (cause or accelerate) chemical reactions. Most strains of E. coli are harmless, but some can cause serious food poisoning.

Bacterial cells cannot live without DHFR, thus this enzyme is the target of many antibiotics. Human cells, and in particular rapidly dividing cells, also use DHFR; drugs that target human DHFR, such as methotrexate, are often used in cancer chemotherapy.

DHFR spurs the conversion of a compound called dihydrofolate (DHF) to a different form, tetrahydrofolate (THF), which is needed by cells for synthesis of DNA. In its chemical reaction, DHFR uses a helper or co-factor, called NADPH. It catalyzes the transfer of a hydride (a negative hydrogen ion) from NADPH to DHF to produce THF. Previous studies by Wright and others have shown that the loops surrounding the active site are flexible, and that one of the loops in particular, called the Met20 loop can adopt two different conformations during the catalytic cycle.

Until now, however, the significance of these motions remained obscure.

Linking Motion to Function

Wright, graduate student Gira Bhabha, and colleagues from both Scripps Research and Pennsylvania State University decided to investigate.

For the new study, the scientists turned to an imaging technique known as nuclear magnetic resonance (NMR) spectroscopy, in combination with X-ray crystallography. Unlike X-ray crystallography, a technique used to determine the structure of proteins in crystals, recently developed NMR methods allow scientists to visualize the motions of proteins in solution. The technique can capture protein motions "in a time scale that is relevant to biology, from microseconds to milliseconds to seconds," said Wright.

To determine the importance of the oscillations, the team set out to make a mutation in the DHFR enzyme that prevented the flexible Met20 loop from moving. To know which amino acids to change, the scientists compared the bacterial DHFR protein sequence to that of the human enzyme, since in the human enzyme, the Met20 loop is more rigid.

Using this approach, the scientists successfully produced a rigidified mutatant E. coli DHFR. When the scientists examined it using X-ray crystallography, they could see the mutant enzyme's structure was almost identical to the wild-type enzyme. However, NMR analysis revealed that the Met20 loop and other parts of the active site were no longer flexible in the mutant.

Significantly, the mutated E. coli enzyme transferred hydride at a rate that was 16-fold slower than that of the wild-type enzyme—a substantial loss in enzyme function.

"We demonstrated that locking down the motion in the active site prevents catalysis," said Wright.

While previous work had indicated that enzymes can exist in different shapes and forms and that changes in enzyme shape enable enzymes to bind to their substrates and co-factors or release the products, "This is the first demonstration that motions play a role in the actual chemistry of a reaction," said Wright.

Clamping Down on the Active Site

The scientists reason that when the E. coli DHFR carries out its chemical reaction, motions in the active site assist in pushing NADPH and DHF closer to one another. This proximity makes the transfer of the hydride from NAPDH to DHF more efficient. If the active site can't move, the molecules are not sufficiently close to one another for the chemical reaction to occur. "We think that the mutations prevent the enzyme from clamping down on the hydride donor and acceptor, so they can no longer get as close to each other as is necessary for efficient catalysis," explained Bhabha.

Taking motion into account when designing drugs to either inhibit or increase enzyme function could result in more effective or more specific drugs. For example, because the motions in the bacterial DHFR differ from those in the human enzyme, this difference might be exploited to design drugs that are specific for the bacterial enzyme. "It might help reduce the serious side effects of drugs that target DHFR," said Wright.

"The idea is to harness these motions in drug design," added Bhabha. "It's a difficult and challenging problem, but it could have huge impact."

In addition to Wright and Bhabha, co-authors for the paper "A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis," include Damian C. Ekiert, Ian A. Wilson, and H. Jane Dyson at Scripps Research, and Jeeyeon Lee, Jongsik Gam, and Stephen J. Benkovic at Pennsylvania State University.

The research was supported by the National Institutes of Health and the Skaggs Institute for Chemical Biology.

About The Scripps Research Institute

The Scripps Research Institute is one of the world's largest independent, non-profit biomedical research organizations. Scripps Research is internationally recognized for its discoveries in immunology, molecular and cellular biology, chemistry, neuroscience, and vaccine development, as well as for its insights into autoimmune, cardiovascular, and infectious disease. Headquartered in La Jolla, California, the institute also includes a campus in Jupiter, Florida, where scientists focus on drug discovery and technology development in addition to basic biomedical science. Scripps Research currently employs about 3,000 scientists, staff, postdoctoral fellows, and graduate students on its two campuses. The institute's graduate program, which awards Ph.D. degrees in biology and chemistry, is ranked among the top ten such programs in the nation. For more information, see www.scripps.edu .

Advanced Molecular Biology Free Ebook Download

2011-04-10T12:21:00.000+05:00

Advanced Molecular Biology emphasises the unifying principles and mechanisms of molecular biology, with frequent use of tables and boxes to summarise experimental data and gene and protein functions. Extensive cross-referencing between chapters is used to reinforce and broaden the understanding of core concepts. This is the ideal source of comprehensive, authoritative and up-to-date information for all those whose work is in the field of molecular biology.

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Apoptosis in Neurobiology Free Ebook Download

2011-04-08T21:38:00.000+05:00

Apoptosis in Neurobiology (Frontiers in Neuroscience)
Publisher: CRC | ISBN: 0849333520 | edition 1998 | File type: PDF | 271 pages | 11,5 mb

The rapid growth of the study of apoptosis-mechanism-driven, regulated cell death-has created an urgent need for reliable documentation of the different approaches to and methods of studying the various aspects of the field. Apoptosis in Neurobiology is an important resource for researchers in this emerging frontier of biomedical study. This volume allows the uninitiated neuroscientist intellectual and practical access to the study of apoptosis, with special consideration to the nervous system. The first section concentrates on conceptual approaches to the study of apoptosis in neurobiology and its significance to the nervous system. The second section provides a user-friendly approach to methods and techniques in the study of apoptosis as applied to neurobiology.

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Methods in Molecular Biology Volume 5: Animal Cell Culture Free Ebook Download

2011-04-08T21:29:00.001+05:00

Animal Cell Culture, the latest volume in Humana's highly successful Methods in Molecular Biology series, provides detailed practical techniques for the culture of a broad spectrum of basic cell cell types. Chapters offer hands-on methods for creating mammalian fibroblastic cell cultures and maintaining culture conditions for epithelial, neuronal, and hematopoietic cells among others. Attention is given to the diversity of culture media and extracellular matrices needed to maintain the differentiated functions of the cultured cells.

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Microarray Method for Genetic Testing

2011-03-29T21:37:00.000+05:00

Genetic testing allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a person's ancestry. Normally, every person carries two copies of every gene, one inherited from their mother, one inherited from their father. The human genome is believed to contain around 20,000 - 25,000 genes. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. Most of the time, testing is used to find changes that are associated with inherited disorders. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.

DNA Sequencing Method: Cycle Sequencing

2011-03-29T10:22:00.000+05:00

To sequence a piece of dna you need 1)a Template DNA 2) a short DNA primer that is complementary to the dna you want to sequence, 3)A enzyme called DNA polymerase,(4) Four nucleotides.(A,C,G,T), To this mix ,we also add a second type of nucleotide; one that has a slightly different chemical formula, These dideoxynucleotides(diddtp) can be recognized by a DNA sequencer.

To start the sequencing reaction this mixture is heated to 96C ,so the template DNA's two complementary strand separates,Then the temperature is lowered, so that the short "primer" sequence finds its complementary sequence in the template DNA.Finally the temperature is raised 60c,this allows the enzyme to bind to the DNA and create a new strand of DNA.

The sequence of this new DNA is complementary to the original DNA strand. The enzyme makes no distinction between dNTPs or didNTPs.each time a didNTP is incorporated, in this case didATP,The synthesis stops. Because billion of DNA molecules are present in the test tube, the strand can be terminated at any position. This results in collection of DNA strands of many different lengths.

The sequencing reaction is transferred from the test tube to a lane of a polyacrylamide gel. The gel is placed into a DNA sequencer for electrophoresis and analysis. The fragments migrate according to size and each is detected as it passes a laser beam at the bottom of the gel. Each type of dideoxynucleotide emits colored light of a characteristic wavelength and is recorded as a colored band on a simulated gel image, and finally computer program interprets the raw data and outputs an electropherogram with colored peaks representing each letter in the sequence.the sequence fragments are sorted out according to the size, starting from the shortest to longest one, the stimulated gel image is read from bottom to top, starting with the smallest fragment, Thus we sequences present in template DNA.

UniSeq DNA Sequencing System

2011-03-23T21:59:00.000+05:00

UniSeq™ is a universal DNA sequencing primer walking technology developed by Nucleics for large scale DNA sequencing applications like whole genome sequencing projects. The UniSeq DNA sequencing library & software offers the following advantages over other sanger DNA sequencing methodologies:

A fast and cost effective universal primer walking approach
Very simple and robust protocol
Compatible with all Sanger DNA sequencing technologies and equipment
Fully adaptable to high throughput, large scale DNA sequencing facilities

The UniSeq DNA sequencing system

The UniSeq DNA sequencing system utilizes a unique methodology to reliably create template specific DNA sequencing primers. The process of generating each UniSeq DNA sequencing primer involves the addition of an "E"- and "T"-oligonucleotide, together with a specifically formulated additive mixture, directly to the sequencing reaction. These oligonucleotides (termed EO and TO) hybridize during the sequencing reaction to produce the template specific primer (S-primer) (Figure 1).

Figure 1. Generation of template specific UniSeq S-primer. The EO hybridizes to the TO and gets extended to form the S-primer. N: degenerated oligonucleotide positions. X: specific positions of variable nucleotide value.
By selecting specific EO and TO oligonucleotides from a small, pre-synthesised library of 768 oligonucleotides, over 131,000 different, template specific primers can be created. This simple combinatorial effect forms the basis for the high specificity and universal applicability of the UniSeq system in DNA sequencing.
The UniSeq system is fully compatible with the common DNA sequencing reagents (e.g. BigDye™ from Applied Biosystems or DYEnamic™ from Amersham Biosciences) and gives excellent results using modern capillary DNA sequencers (Figure 2).

Figure 2. UniSeq reaction sequencing trace. Two hundred nanograms of plasmid DNA was sequenced with BigDye vers.3.1 and UniSeq primers EO 18 and TO 447. The DNA sequencing reaction was purified by ethanol precipitation and analyzed on an ABI 3700 DNA sequencer.
The UniSelect™ DNA sequencing software is provided for the automated and optimized selection of the optimal EO & TO primer pairs for each DNA template. The UniSelect software also supplies an automated interface to control the robotic pipetting of the UniSeq oligonucleotides, DNA templates and other required sequencing reagents.
To aid the finishing of Whole Genome Sequencing (WGS) projects, an additional software package (UniFinish™) is available. UniFinish is able to parse ACE format assembly files and select the EO/TO oligonucleotides, together with the appropriate DNA templates, required to close non-physical DNA sequence gaps in the genome assembly.

Feature and benefits of UniSeq

Genomes are finished faster

The UniSeq DNA sequencing system provides a competitive alternative to DNA sequencing with custom synthesized oligonucleotides. While many advances have been made in the automation of oligonucleotide synthesis, this process is still complex (eg. requiring high maintenance machinery) and slow (several hours per synthesis). These limitations have become extremely critical for high throughput DNA sequencing facilities where modern capillary DNA sequencers have reduced the separation time to one to two hours. The generation of specific DNA sequencing primers by the UniSeq process during the DNA sequencing reaction step (i.e. no time-costs) breaks the current bottleneck imposed by custom oligonucleotide synthesis and allows for the most efficient utilization of existing DNA sequencing instruments and other equipment.

Lower per finished base costs

Currently, two major strategies are used in WGS – Random Shotgun DNA Sequencing (RSS) and Primer Walking DNA Sequencing (PWS). Most WGS projects currently employ RSS, especially in early stages of projects. However, RSS requires generating a large amount of redundant DNA sequence (commonly 6 to 15 times the genome size) for assembly purposes. The alternative PWS strategy requires relatively little redundant DNA sequence data, however, it requires a large numbers of custom made oligonucleotide primers. The high costs (US$3 to 5 per primer), together with the required synthesis delay, have prevented the general adoption of PWS strategy in WGS Genomics projects.
UniSeq provides the advantages of both WGS strategies. It offers the speed and simplicity of the RSS approach, while providing the data efficiency inherent with the PWS approach. Computer simulations and limited trials have shown that UniSeq DNA sequencing system offers cost and time savings of greater than 80% over current WGS approaches.

Increased DNA sequencing flexibility

Nucleics has extensively tested UniSeq in house in a number of general DNA sequencing and WGS projects. In addition, Nucleics has formulated novel strategies for the easy and smooth implementation of UniSeq into industrial scale DNA sequencing facilities. We also offer consulting services to help integrate UniSeq into new or existing DNA sequencing facilities.

DNA Sequencing: Shotgun Sequencing

2011-03-23T21:51:00.000+05:00

Shotgun Sequencing was first developed by Craig Ventor in 1996. He developed it as he was working in the Genome Research Institute. It was him who also made it popular. He then went on to start his Celera Corporation with the sole mission and goal of doing the sequencing of mainly the human genome in as little as three years. But of course, some say that in genetics, the shotgun sequencing was in fact first developed by the double Nobel laureate, Fred Sanger, in the 1970s .
Shotgun Sequencing is a DNA sequencing method that involves the physically breaking down of a long stretch of DNA into very small fragments; about 2,000 base-pair. These fragments are then cloned, sequenced and also assembled with the use of computer analysis.
Also known in other circles and by other people as shotgun cloning, this method is said to be one of the harbinger technologies which is mainly responsible for bringing about what we now have as full genome sequencing. The sequencing of the human genome was done by Craig Ventor’s Celera Corporation as well as by the Human Genome Project. They used a map-based sequencing while Craig’s Ventor’s Celera used shotgun sequencing.
Today, however, Craig Ventor’s Shotgun Sequencing is the preferred system and method for doing other types of genome sequencing. And as science and technology improves, it’s clearly obvious that more things will be discovered that will alter or improve the method, for the better. For example, there is now the method called the next-generation sequencing, which is said to result in high coverage than Craig’s. As they say, only time will tell what the future has for this and other methods yet unravelled because man is discovering newer things each and every day. And as many continue to share what they know with others, better methods will obviously be discovered.

Lehninger's Principles of Biochemistry - 5th Edition Ebook Download

2011-03-23T16:12:00.000+05:00

In the Fifth Edition, authors Dave Nelson and Mike Cox combine the best of the laboratory and best of the classroom, introducing exciting new developments while communicating basic principles through a variety of new learning tools—from new in-text worked examples and data analysis problems to the breakthrough eBook, which seamlessly integrates the complete text and its media components.

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Separation of DNA Fragments Using PAGE Method

2011-03-20T15:24:00.000+05:00

This method is able to separate DNA fragments with the size of as small as 10 bp and up to 1 kb with the resolution of as little as 1 bp. While agarose gel electrophoresis is only able to separate DNA fragments with the bigger size that PAGE does or in the size range of 100 nucleotides to around 10 – 15 kb.

Materials:

Gel apparatus: Many designs of apparatus are commercially available. The gel is poured between two vertical plates held apart by spacers

The plates should be cleaned thoroughly and then wiped with ethanol. To help ensure that the gel only sticks to one plate when the apparatus is disassembled, apply silicon to one of the gel plates. This is easily done by wiping the plate with a tissue soaked in dimethyl dichlorosilane solution and then washing the plate in distilled water followed by ethanol. If the plates are baked at 100^oC for 30 min, the siliconization will last four to five gel runs.
Deionized H₂O: Autoclaved water is not necessary for the gel mix or running buffer, but it should be used for diluting samples and purification from gel slices.
l0x TBE: 108 g of Trizma base (Tris), 55 g of boric acid, and 9.3 g of ethylenediaminetetraacetic acid (EDTA) (disodium salt). Make up to I L solution with deionized H₂O, which should be discarded when a precipitate forms.
Acrylamide stock: 30% acrylamide, 1% N,N'-methylene bisacrylamide. Store at 4^oC. This is available commercially, or it can be made by dissolving acrylamide and bisacrylamide in water, which should be filtered. Acrylamide is a neurotoxin and therefore must be handled carefully. Gloves and a mask must be worn when weighing out.
APS: 10% Ammonium persulphate (w/v). This can be stored at 4^oC for 1-2 months.
TEMED: N,N,N',N'-tetramethyl-l,2-diaminoethane. Store at 4^oC.
5X sample buffer: 15% Ficoll solution, 2.5X TBE, 0.25% (w/v) xylene cyanol and 0.025% (w/v) bromophenol blue.
Ethidium bromide: A l0-mg/mL solution. Ethidium bromide is a potent mutagen and should be handled with care. Store at 4^oC in the dark.

Methods:

For 50 mL, enough for a 18 x 14 x 0.15 cm gel, mix l0x TBE, acrylamide, H₂O, and APS as described in Table below.

Just prior to pouring, add 50 microliters of TEMED and mix by swirling.
Immediately pour the gel mix between the gel plates and insert the gel comb. Leave to set; this takes about 30 min.
Fill the gel apparatus with 0.5X TBE and remove the comb. Use a syringe to wash out the wells, this may take multiple washes. It is important to remove as much unpolymerized acrylamide as possible because this impairs the running in of the samples

If you are separating very small fragments, e.g., less than 50 bp, the gel should be prerun for 30 min, as this elevates the resolution problem experienced with fragments running close to the electrophoresis front..
Add 0.2 volume of 5x sample buffer to each sample, usually in 10-20 microliters of TE, water, or enzyme buffer. Mix and spin the contents to the bottom of the tube

High-salt buffers (above 50 mM NaCl) will affect sample mobility and tend to make bands collapse. In this case, salt should be removed by ethanol precipitation..
Load the samples on the gel and run at 200-300 V (approximately 10 V/cm) until the bromophenol blue band is two-thirds of the way down the gel; this takes about 2.5 h

Do not run the gel faster than 10 V/cm, as this will cause the gel to overheat, affecting the resolution. The gel can be run more slowly, e.g., 75 V will run overnight.
Disassemble the gel apparatus and place the gel to stain in I mg/mL of ethidium bromide for approximately 30 min. View the stained gel on a transilluminator.

Happy separating DNA fragments.

Purification Of Plasmid DNA

2011-03-14T22:36:00.000+05:00

After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes. In order to purify plasmid DNA after the isolation process, any residual RNA and contaminating protein are removed. This purification step involves two main steps, which are, first, removing residual RNA by using RNase in order to digest RNA and, second, extract contaminating protein using organic solvents, phenol-chloroform.

Materials:

RNase A: Make up as a solution in water at 10 mg/mL, Heat for 10 min in a boiling water bath or heating block to eliminate any DNase activity. Aliquot and store at -20^oC.
0.4 M Ammonium acetate.
Chloroform= A 24: 1 mix of chloroform and isoamyl alcohol. Store at 4^oC.
Phenol/chloroform= 25:24: 1 mix of TE-equilibrated phenol, chloroform, and isoamyl alcohol. Store at 4^oC.
100% Ethanol.
Sterile wooden toothpicks.

Methods:

Add 50 microliters of 4 M ammonium acetate containing 200 micrograms/mL RNase A to each miniprep and incubate it at room temperature for 20 min.
Add 100 microliters of phenol/chloroform to each DNA preparation.
Vortex briefly and centrifuge at high speed for 2 min in a microfuge. Remove the top layer containing the DNA and place it in a new sterile tube.
Add 100 microliters of chloroform to each tube.
Vortex briefly and centrifuge at high speed in a microfuge for 2 min. Remove the DNA in the top layer and place it in a second sterile tube.

For phenol/chloroform extractions avoid removing material from the interface.
Add 200 microliters of 100% ethanol to each tube.
Shake briefly to precipitate the DNA and centrifuge at high speed for 5 min at room temperature.

Done. Now you can extract, isolate, and purify Plasmid DNA using methods which I had described in this blog. Hopefully those can be useful for you.

The Vertical Farm

2011-03-11T16:38:00.000+05:00

The Problem

By the year 2050, nearly 80% of the earth's population will reside in urban centers. Applying the most conservative estimates to current demographic trends, the human population will increase by about 3 billion people during the interim. An estimated 10⁹ hectares of new land (about 20% more land than is represented by the country of Brazil) will be needed to grow enough food to feed them, if traditional farming practices continue as they are practiced today. At present, throughout the world, over 80% of the land that is suitable for raising crops is in use (sources: FAO and NASA). Historically, some 15% of that has been laid waste by poor management practices. What can be done to avoid this impending disaster?

A Potential Solution: Farm Vertically

The concept of indoor farming is not new, since hothouse production of tomatoes, a wide variety of herbs, and other produce has been in vogue for some time. What is new is the urgent need to scale up this technology to accommodate another 3 billion people. An entirely new approach to indoor farming must be invented, employing cutting edge technologies. The Vertical Farm must be efficient (cheap to construct and safe to operate). Vertical farms, many stories high, will be situated in the heart of the world's urban centers. If successfully implemented, they offer the promise of urban renewal, sustainable production of a safe and varied food supply (year-round crop production), and the eventual repair of ecosystems that have been sacrificed for horizontal farming.

It took humans 10,000 years to learn how to grow most of the crops we now take for granted. Along the way, we despoiled most of the land we worked, often turning verdant, natural ecozones into semi-arid deserts. Within that same time frame, we evolved into an urban species, in which 60% of the human population now lives vertically in cities. This means that, for the majority, we humans are protected against the elements, yet we subject our food-bearing plants to the rigors of the great outdoors and can do no more than hope for a good weather year. However, more often than not now, due to a rapidly changing climate regime, that is not what follows. Massive floods, protracted droughts, class 4-5 hurricanes, and severe monsoons take their toll each year, destroying millions of tons of valuable crops. Don't our harvestable plants deserve the same level of comfort and protection that we now enjoy? The time is at hand for us to learn how to safely grow our food inside environmentally controlled multistory buildings within urban centers. If we do not, then in just another 50 years, the next 3 billion people will surely go hungry, and the world will become a much more unpleasant place in which to live.

Advantages of Vertical Farming

Year-round crop production; 1 indoor acre is equivalent to 4-6 outdoor acres or more, depending upon the crop (e.g., strawberries: 1 indoor acre = 30 outdoor acres)
No weather-related crop failures due to droughts, floods, pests
All VF food is grown organically: no herbicides, pesticides, or fertilizers
VF virtually eliminates agricultural runoff by recycling black water
VF returns farmland to nature, restoring ecosystem functions and services
VF greatly reduces the incidence of many infectious diseases that are acquired at the agricultural interface
VF converts black and gray water into potable water by collecting the water of
evapotranspiration
VF adds energy back to the grid via methane generation from composting non-edible
parts of plants and animals
VF dramatically reduces fossil fuel use (no tractors, plows, shipping.)
VF converts abandoned urban properties into food production centers
VF creates sustainable environments for urban centers
VF creates new employment opportunities
We cannot go to the moon, Mars, or beyond without first learning to farm indoors on
earth
VF may prove to be useful for integrating into refugee camps
VF offers the promise of measurable economic improvement for tropical and subtropical
LDCs. If this should prove to be the case, then VF may be a catalyst in helping to reduce or even reverse the population growth of LDCs as they adopt urban agriculture as a strategy for sustainable food production.
VF could reduce the incidence of armed conflict over natural resources, such as water
and land for agriculture

Urban Farming Grows Up

2011-03-11T16:28:00.001+05:00

Urban Farming’s mission is to create an abundance of food for people in need by planting, supporting and encouraging the establishment of gardens on unused land and space while increasing diversity, raising awareness for health and wellness, inspiring and educating youth, adults and seniors to create an economically sustainable system to uplift communities around the globe.

INDIRECT ELISA

2011-03-04T16:46:00.000+05:00

INDIRECT ELISA :

INDIRECT ELISA The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies).

INDIRECT ELISA :

INDIRECT ELISA Advantages of indirect detection Wide variety of labeled secondary antibodies are available commercially. Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. Immunoreactivity of the primary antibody is not affected by labeling. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.

DIRECT ELISA

2011-03-04T16:39:00.000+05:00

DIRECT ELISA :

DIRECT ELISA The direct ELISA uses the method of directly labeling the antibody itself. Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point.

DIRECT ELISA :

DIRECT ELISA Advantages of Direct Detection Quick methodology since only one antibody is used. Cross-reactivity of secondary antibody is eliminated. Disadvantages of Direct Detection Immunoreactivity of the primary antibody may be reduced as a result of labeling. Labeling of every primary antibody is time-consuming and expensive. No flexibility in choice of primary antibody label from one experiment to another. Little signal amplification.

COMPETITIVE ELISA

2011-03-04T16:32:00.000+05:00

COMPETITIVE ELISA :
COMPETITIVE ELISA In this Unlabeled antibody is incubated in the presence of its antigen. These bound antibody/antigen complexes are then added to an antigen coated well. The plate is washed unbound antibody is removed. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.

ELISA : Enzyme-Linked ImmunoSorbent Assay

2011-03-04T16:23:00.000+05:00

How the Test is Performed

Blood is typically drawn from a vein, usually from the inside of the elbow or the back of the hand. The site is cleaned with germ-killing medicine (antiseptic). The health care provider wraps an elastic band around the upper arm to apply pressure to the area and make the vein swell with blood.
Next, the health care provider gently inserts a needle into the vein. The blood collects into an airtight vial or tube attached to the needle. The elastic band is removed from your arm.
Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding.
In infants or young children, a sharp tool called a lancet may be used to puncture the skin and make it bleed. The blood collects into a small glass tube called a pipette, or onto a slide or test strip. A bandage may be placed over the area if there is any bleeding.
The sample is sent to a laboratory where the targeted antibody (or antigen) is linked to an enzyme. If the target substance is in the sample, the test solution turns a different color.

How to Prepare for the Test

No special preparation is needed.

How the Test Will Feel

When the needle is inserted to draw blood, some people feel moderate pain, while others feel only a prick or stinging sensation. Afterward, there may be some throbbing.

Why the Test is Performed

This test is often used to see if you have been exposed to viruses or other substances that cause infection. It is often used to screen for current or past infections.

Normal Results

Normal values depend on the type of substance being identified. Normal value ranges may vary slightly among different laboratories. Talk to your doctor about the meaning of your specific test results.

What Abnormal Results Mean

Abnormal values depend on the type of substance being identified. In some people, a positive result may be normal.

Risks

Veins and arteries vary in size from one patient to another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than from others.
Other risks associated with having blood drawn are slight but may include:

Excessive bleeding
Fainting or feeling light-headed
Hematoma (blood accumulating under the skin)
Infection (a slight risk any time the skin is broken)

Alternative Names

Enzyme-linked immunoassay; EIA

References

Ashihara Y, Kasahara Y, Nakamura RM. Immunoassay and immunochemistry. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 21st ed. Philadelphia, Pa: Saunders Elsevier; 2006:chap 43.

Plasmid DNA Extraction Using Alkaline Lysis Method

2011-02-13T14:17:00.000+05:00

Plasmids can be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. One commonly used technique developed by Birnboim and Doly involves alkaline lysis. This method essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol.
Materials:

Luria Bertani (LB) broth bacteria culture medium: 1% Tryptone, 0.5% yeast extract, 200 mM NaCl. Sterilize by autoclaving in suitable aliquots. In order to ensure retention of the plasmid, media should be supplemented with the appropriate antibiotic(s).
1.5 mL Microfuge tubes.
Sterile tubes: Must have a volume of at least 10 mL to ensure good aeration.
Lysis solution: 200 mM NaOH, 1% SDS. Store at room temperature.
Resuspension solution: 50 mM glucose, 50 mM Tris-HCl, pH 8.0, 10 mM ethylene diamine tetraacetic acid (EDTA). Keep at 4^oC to prevent growth of contaminants.
Potassium acetate (neutralizing solution): 3 M potassium/5 M acetate. For 100 mL, take 29.4 g of potassium acetate, add water to 88.5 mL, and 11.5 mL of glacial acetic acid. Store at room temperature.
TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
Isopropanol.
70% Ethanol.

Methods:

Take a number of separate sterile tubes and place 2 mL of L-broth into them. Inoculate from individual bacterial 37°C overnight with shaking.
Transfer each culture to a labeled 1.5-mL Eppendorf tube, and centrifuge for 30 s at high speed in the microfuge.
Here, a tight creamy pellet may be seen.
Decant the supernatant and place tubes in a rack vertically for 5-10 s. Remove any of the remaining liquid by aspiration with a fine Pasteur pipet.
Add 100 microliters of resuspension solution into each tube, close the lids, and resuspend the bacteria in each tube by shaking or vortexing to dissociate the bacterial pellet.
To each tube add 200 microliters of lysis solution and mix by inverting the tube several times.
The solution should quickly turn transparent and become more viscous indicating bacterial
lysis has taken place.
Allow at least 2-3 min for lysis to take place and leave the tubes to stand for 60 s before opening. This will allow the liquid to return to the bottom of the tube.
To each tube add 150 microliters of neutralizing solution and invert the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.
Centrifuge the tubes for 2-5 min at full speed in a microfuge.
Place new sterile tubes into a rack, label them, and add 250 ~ of isopropanol to each tube.
Remove the tubes from the microfuge, being careful not to disturb the precipitate.
Remove the supernatant with a 1-mL pipet, avoiding the white precipitate as much as possible.
Some of the precipitate may float, so it is critical to use a pipet and disposable tips to
recover the supernatant rather than pouring it.
Transfer the liquid phase into the new set of labeled tubes containing the isopropanol.
Vortex the tubes for 5-10 s and centrifuge the tubes in the microfuge for 30 s at high speed. The plasmid DNA precipitates as a white pellet.
Decant the supernatant and wash the pellets by adding 750 mL of 70% ethanol, vortex briefly, and centrifuge at high speed for 30 s.
Decant the ethanol, and centrifuge again for 10 s to collect the remaining ethanol at the bottom of the tubes. Carefully aspirate the remaining ethanol and leave the tubes to air dry on the bench for 5 min.
Dispense 50 microliter of TE into each tube, and resuspend the pellet
It is not advisable to vortex, as this may lead to DNA shearing. The sample is best left for
3-5 min with occasional finger flicking of the tube.
Take 10 microliters of the resuspended pellet and analyze by agarose gel electrophoresis (see on my posting entitled Agarose Gel Electrophoresis of Nucleic Acids

Size Exclusion Chromatography

2011-02-07T22:05:00.000+05:00

Size Exclusion Chromatography (also known as gel filtration) separates molecules based on molecular size. This chromatography can be applied using resins or membrane. With membranes, the smaller molecules pass through while the larger molecules (above a certain size cut-off) are held above the membrane. With resins, the larger molecules pass/flow through the resin and are collected first while the smaller molecules take longer to flow through because these smaller particles get held up within the pores of the resins. Therefore, with resins, the sample passes through the resin in decreasing molecular weight. Common size exclusion applications include concentration, fractionation, desalting and buffer exchange.

Size exclusion is one of the easiest chromatography methods to perform because samples are processed using an isocratic elution. In its analytical form, size exclusion can distinguish between molecules (e.g. proteins) with a molecular weight difference of less than a factor of two times. In this application, the porosity of the filtration media to be used is selected to provide high resolution in the molecular weight range of interest.

How to Make and Run an Agarose Gel (DNA Electrophoresis)

2011-01-19T00:43:00.000+05:00

Gel electrophoresis is a method that separates (based on size, electrical charge and other physical properties) macromolecules such as nucleic acids or proteins.
The electrophoresis term is used to describe the migration of charged particle under the influence of an electric field. Thus gel electrophoresis refers is the technique in which molecules are forced across a span of gel, motivated by an electrical current. On either end of the gel there are activated electrodes that provide the driving force. Therefore a molecule's properties especially the possession of ionisable groups, determine how rapidly an electric field can move the molecule through a gelatinous medium.
One very important application for gel electrophoresis is in DNA Technology. Biotechnology has for thousands of years been used by people who have used yeast to make flour into bread and grapes into wine. We are now using biotechnology to study the basic processes of life, diagnose illnesses, and develop new treatments for diseases. Some of the tools of biotechnology are natural components of cells. For example restriction enzymes are made by bacteria to protect themselves from viruses. They inactivate the viral DNA by cutting it in specific places. DNA ligase is an enzyme that exists in all cells and is responsible for joining together strands of DNA. Restriction enzymes can be used to cut DNA at specific sequences called recognition sites. They then rejoin the cut strands with DNA ligase to new combinations of genes. Recombinant DNA sequences contain genes from two or more organisms.
This technique has allowed researchers to gain the ability to diagnose diseases such as sickle cell anemia, cystic fibrosis, and Huntington's chorea early in the course of the disease. Many researchers are also applying the techniques of biotechnology to find new treatments for genetic diseases.
DNA technology has triggered research advances in almost all fields of biology. Currently hundreds of useful products are produced by genetic engineering. It has become routine to combine genes from different sources, usually different species--in test tubes, and then transfer this recombinant DNA into living cells where it can be replicated and expressed.
The most important achievements resulting from recombinant DNA technology have been advances in our basic understanding of eukaryotic molecular biology. For example, only through the use of gene-splicing techniques have the details of eukaryotics gene arrangement and regulation been opened to experimental analysis.
Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study. One use is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity.
Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid.